CIF130 inhibits cell cycle progression

ABSTRACT

A human gene termed CIF130 and its expression products can alter the spatial or temporal patterns of mitosis or cell cycle progression of a human cell. Methods of treating disorders involving alterations in the regulation of mitosis or cell cycle progression utilize the gene and its expression product. Genes whose expression is dependent upon CIF130 expression can be identified.

[0001] This application claims the benefit of co-pending provisionalapplications Serial No. 60/089,198 filed Jun. 12, 1998, and Ser. No.60/111,636 filed Dec. 9, 1998, which are incorporated herein byreference.

TECHNICAL AREA OF THE INVENTION

[0002] The invention relates to the area of cell cycle progression. Moreparticularly, the invention relates to proteins which regulate cellcycle progression.

BACKGROUND OF THE INVENTION

[0003] Alterations in the regulation of cell cycle progression play animportant role in diseases such as neoplasia and anemia. Manipulation ofgenes involved in regulating the cell cycle can be used to prevent ortreat these diseases Detections of mutations in cell-cycle regulatorygenes can also be used to detect neoplastic cells and geneticpredispositions to neoplasias. Thus, there is a need in the art for theidentification of cell cycle regulator genes which can be used inmethods of diagnosing, prognosing, and treating neoplasia and otherdiseases in humans and other mammals.

SUMMARY OF THE INVENTION

[0004] It is an object of the invention to provide reagents and methodsfor regulating mitosis or cell cycle progression in human cells and fortreating disorders related to alterations in cell cycle progression.These and other objects of the invention are provided by one or more ofthe embodiments described below.

[0005] One embodiment of the invention is an isolated and purified humanCIF130 protein comprising an amino acid sequence which is at least 85%identical to the amino acid sequence shown in SEQ ID NO: 2. Percentidentity is determined using a Smith-Waterman homology search algorithmusing an affine gap search with a gap open penalty of 12 and a gapextension penalty of 1.

[0006] Another embodiment of the invention is an isolated polypeptidecomprising at least 17 contiguous amino acids as shown in SEQ ID NO: 2.

[0007] Yet another embodiment of the invention is a CIF130 fusionprotein comprising a first protein segment and a second protein segmentfused together by means of a peptide bond. The first protein segmentconsists of at least 17 contiguous amino acids of a human CIF130 proteinas shown in SEQ ID NO: 2.

[0008] Even another embodiment of the invention is a preparation ofantibodies which specifically bind to a human CIF130 protein having anamino acid sequence as shown in SEQ ID NO: 2.

[0009] Still another embodiment of the invention is a cDNA moleculewhich encodes a human CIF130 protein having an amino acid sequence whichis at least 85% identical to SEQ ID NO: 2. Percent identity isdetermined using a Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 1.

[0010] A further embodiment of the invention is a cDNA molecule whichencodes at least 17 contiguous amino acids of SEQ ID NO 2.

[0011] Another embodiment of the invention is a cDNA molecule comprisingat least 12 contiguous nucleotides of SEQ ID NO: 1.

[0012] Still another embodiment of the invention is a cDNA moleculewhich is at least 85% identical to the nucleotide sequence shown in SEQID NO: 1. Percent identity is determined using a Smith-Waterman homologysearch algorithm as implemented in a MPSRCH program using an affine gapsearch with a gap open penalty of 12 and a gap extension penalty of 1.

[0013] Even another embodiment of the invention is an isolated andpurified subgenomic polynucleotide comprising a nucleotide sequencewhich hybridizes to SEQ ID NO: 1 after washing with 0.2×SSC at 65° C.The nucleotide sequence encodes a CIF130 protein having the amino acidsequence of SEQ ID NO: 2.

[0014] Yet another embodiment of the invention is a construct comprisinga promoter and a polynucleotide segment encoding at least 17 contiguousamino acids of a human CIF130 protein as shown in SEQ ID NO: 2. Thepolynucleotide segment is located downstream from the promoter.Transcription of the polynucleotide segment initiates at the promoter.

[0015] Even another embodiment of the invention is a host cellcomprising a construct which comprises a promoter and a polynucleotidesegment encoding at least 17 contiguous amino acids of a human CIF130protein having an amino acid sequence as shown in SEQ ID NO: 2.

[0016] A further embodiment of the invention provides a homologouslyrecombinant cell. The homologously recombinant cell incorporates a newtranscription initiation unit. The new transcription initiation unitcomprises an exogenous regulatory sequence, an exogenous exon, and asplice donor site. The transcription initiation unit is located upstreamof a coding sequence of a CIF130 gene as shown in SEQ ID NO: 1. Theexogenous regulatory sequence directs transcription of the codingsequence of the CIF130 gene.

[0017] Another embodiment of the invention provides a method to aid inthe diagnosis or prognosis of neoplasia in a human. Expression of afirst CIF130 gene in a first tissue of a human suspected of beingneoplastic is compared with expression of a second CIF130 gene in asecond tissue of a human which is normal. The second CIF130 gene has thecoding sequence shown in SEQ ID NO: 1. Decreased expression of the firstCIF130 relative to expression of the second CIF130 gene indicatesneoplasia in the first tissue.

[0018] Another embodiment of the invention provides a method to aid inthe diagnosis or prognosis of neoplasia in a human. A first human CIF130gene, mRNA, or protein in a first tissue suspected of being neoplasticis compared with a second human CIF130 gene, mRNA, or protein in asecond tissue which is normal. The second CIF130 gene has the codingsequence shown in SEQ ID NO: 1. A difference between the first andsecond CIF130 genes, mRNAs, or proteins indicates neoplasia in the firsttissue.

[0019] Yet another embodiment of the invention provides a method to aidin detecting a genetic predisposition to neoplasia in a human. A CIF130gene, mRNA, or protein in a fetal tissue of a human is compared with awild-type human CIF130 gene, mRNA, or protein. The wild-type CIF130 genehas the coding sequence shown in SEQ ID NO: 1. A difference between theCIF130 gene, mRNA, or protein in the fetal tissue of the human and thewild-type human CIF130 gene, mRNA, or protein indicates a geneticpredisposition to neoplasia in the human.

[0020] Still another embodiment of the invention provides a method ofscreening test compounds for the ability to interfere with the bindingof a CIF130 protein to a CIF150/hTAF_(II)150 protein. A test compound iscontacted with at least a CIF150/hTAF_(II)150-binding domain of a CIF130protein as shown in SEQ ID NO: 2 and at least a CIF130-binding domain ofa CIF150/hTAF_(II)50 protein as shown in SEQ ID NO: 4. TheCIF130-binding domain binds to the CIF150/hTAF_(II)150-binding domain inthe absence of the test compound. The amount of at least one of theCIF130- or CIF150/hTAF_(II)150-binding domains which is bound or unboundin the presence of the test compound is determined. A test compoundwhich decreases the amount of bound CIF130- orCIF150/hTAF_(II)150-binding domain or which increases the amount unboundCIF130- and CIF150/hTAF_(II)50-binding domains is a potential inducer ofmitosis or cell cycle progression.

[0021] Another embodiment of the invention provides a method ofscreening test compounds for the ability to interfere with the bindingof a CIF 130 protein to a CIF150/hTAF_(II)150 protein. A cell iscontacted with a test compound. The cell comprises two fusion proteins.A first fusion protein comprises (1) a CIF150/hTAF_(II)150-bindingdomain of a CIF130 protein as shown in SEQ ID NO: 2 and (2) either a DNAbinding domain or a transcriptional activating domain. A second fusionprotein comprises a CIF130-binding domain of a CIF150/hTAF_(II)150protein as shown in SEQ ID NO: 4. The CIF130-binding domain binds to theCIF150/hTAF_(II)150-binding domain. If the first fusion proteincomprises a DNA binding domain, then the second fusion protein comprisesa transcriptional activating domain. If the first fusion proteincomprises a transcriptional activating domain, then the second fusionprotein comprises a DNA binding domain. The interaction of the first andsecond fusion proteins reconstitutes a sequence-specific transcriptionactivating factor. The cell also comprises a reporter gene comprising aDNA sequence to which the DNA binding domain specifically binds.Expression of the reporter gene is measured. A test compound whichdecreases the expression of the reporter gene is a potential inducer ofmitosis or cell cycle progression.

[0022] The present invention thus provides the art with reagents andmethods of regulating mitosis or cell cycle progression of human cellsand of treating disorders associated with alterations in cell cycleprogression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1. FIG. 1 shows nucleotide coding sequences of the CIF130gene (SEQ ID NO: 1) and translated amino acid sequences of forms ofCIF130 protein. Full length CIF130 alternate start codons and a 38basepair insert are indicated in the Figure. cDNA translation is SEQ IDNO: 2.

[0024]FIG. 2. FIG. 2 provides the DNA sequence of CIF150 (SEQ ID NO: 3).

[0025]FIG. 3. FIG. 3 provides the amino acid sequence of CIF150 (SEQ IDNO: 4).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] It is a discovery of the present invention that a CIF130 geneproduct binds to and negatively regulates CIF150/hTAF_(II)150 activity.CIF150/hTAF_(II)150 is required for a human cell to enter mitosis.Kaufmann et al., Mol. Cell. Biol. 18(1): 233-39 (1998). In particular,CIF150/hTAF_(II)150 is an essential cofactor for TF_(II)D-dependenttranscription from promoters containing initiator elements, such as theadenovirus major late promoter. CIF150/hTAF_(II)150 also directlyinteracts with the transcription factor hTAF_(II)135 and stimulatescyclin A and B1 expression. A nucleotide sequence encodingCIF150/hTAF_(II)150 is shown in SEQ ID NO: 3. The amino acid sequence ofCIF150/hTAF_(II)150 is shown in SEQ ID NO: 4.

[0027] CIF130 binds to and negatively regulates CIF150/hTAF_(II)150.CIF130 has sequence homology to ATP-dependent RNA helicases (DEAD-boxproteins), which block entry into mitosis in Schizosaccharomyces pombe.CIF130 is implicated in promoter clearance and processing of precursorRNA, particularly in the release of spliced mRNA from the spliceosomeafter splicing of precursor RNA is complete. Thus, CIF130 provides apotential link between transcriptional and translational regulation ofgene expression, by modifying the structure of the nascent RNA. Inaddition, CIF130 mRNA is differentially expressed in tumors, such ashuman brain, uterine, Fallopian tube, and ovarian tumors, compared tonormal tissues.

[0028] CIF130 protein can exist in a number of forms. One form of CIF130has the amino acid sequence shown in SEQ ID NO: 2. Other forms canresult from translation of the CIF130 gene starting at alternate startcodons and/or from inclusion or omission of a 38 basepair insertion inthe CIF130 mRNA (FIG. 1). Protein variants of forms of CIF130 proteinare also included in the invention. Protein variants can be naturally ornon-naturally occurring. Naturally occurring CIF130 variants are thosewhich are found in humans or other species and which comprise amino acidsequences which are substantially identical to the amino acid sequenceshown in SEQ ID NO.2 or to shorter forms of this amino acid sequence, asindicated in FIG. 1, with or without the 12 amino acid insert encoded bythe optional 38 basepair insert. Non-naturally occurring CIF130 variantswhich differ by as much as, for example, four amino acids and retainsubstantially the same biological activities as naturally occurringCIF130 variants are also included here.

[0029] Preferably, naturally or non-naturally occurring protein variantshave amino acid sequences which are at least 85%, 90%, or 95% identicalto the amino acid sequence shown in SEQ ID NO: 2 or to a shorter portionof SEQ ID NO: 2 as shown in FIG. 1 and have similar biologicalproperties, including the ability to bind to CIF150/hTAF_(II)150 and toinhibit mitosis or cell cycle progression. More preferably, themolecules are 98% or 99% identical. Percent sequence identity between aputative human CIF130 variant and the amino acid sequence of SEQ ID NO:2 or to a shorter portion of SEQ ID NO: 2 as shown in FIG. 1 isdetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 1. The Smith-Waterman homology search algorithm is taught inSmith and Waterman, Adv. Appl. Math. (1981) 2:482-489.

[0030] Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, such as DNASTAR software. Preferably, the amino acid changesin CIF130 variants or derivatives are conservative amino acid changes,i.e., substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cystine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

[0031] It is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on thebinding properties of the resulting CIF130 variant, especially if thereplacement does not involve an amino acid at the CIF150/hTAF_(II)150binding site of CIF130. Variants of CIF130 proteins bind toCIF150/hTAF_(II)150 and inhibit mitosis or cell cycle progression.Properties and functions of CIF130 variants are of the same type as aCIF130 protein having the amino acid sequence shown in SEQ ID NO: 2 or ashorter version of SEQ ID NO: 2 as shown in FIG. 1 although theproperties and functions may differ in degree. Whether an amino acidchange results in a functional CIF130 protein or polypeptide variantable to bind to CIF150/hTAF_(II)150 and inhibit mitosis or cell cycleprogression can readily be determined using, for example, in vitro DNAbinding assays, as taught in Kaufmann & Smale, 1994, Genes Devel.8:821-29 and Kaufmann et al, 1996, Genes Devel. 10:873-86, or byassaying the ability of the CIF130 protein or polypeptide variant toinhibit cell cycle progression, as described below.

[0032] CIF130 polypeptides or polypeptide variants differ in length fromnatural CIF130 polypeptides and contain 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000 or more amino acids of a CIF130 proteinor variant in the same primary order as found in a CIF130 protein orvariant obtained from a natural source. Polypeptide molecules havingsubstantially the same amino acid sequence as CIF130 but possessingminor amino acid substitutions which do not substantially affect theability of the CIF130 polypeptide variants to interact withCIF130-specific molecules, such as CIF150/hTAF_(II)150 or antibodieswhich specifically bind to CIF130, are within the definition of CIF130.CIF130 variants include glycosylated forms, aggregative conjugates withother molecules, and covalent conjugates with unrelated chemicalmoieties.

[0033] CIF130 variants of the invention also include allelic variants,species variants, and muteins. Truncations or deletions of regions whichdo not affect the properties or functions of CIF130 described above arealso variants of CIF130 Covalent variants can be prepared by linkage offunctionalities to groups which are found in the amino acid chain or atthe N- or C-terminal residue, as is known in the art.

[0034] A subset of mutants, called muteins, is a group of polypeptideswith the non-disulfide bond participating cysteines substituted with aneutral amino acid, generally, with serines. These mutants may be stableover a broader temperature range than CIF130 See Market al., U.S. Pat.No. 4,959,314.

[0035] Naturally occurring CIF130 proteins or polypeptides can bepurified from human cells or cell lines, such as HeLa or NIH 3T3 cells,by methods known in the art. CIF130 copurifies with CIF150/hTAF_(II)150;thus, the initial purification steps for each protein are the same.Preferably, CIF130 is purified from HeLa cell extracts (Dignam et al.,1983, Nucl. Acids Res. 11:1475-89) by Ni affinity chromatography, asdescribed in Example 1. Purified CIF150/hTAF150 and CIF130 proteins canbe visualized by sodium dodecyl sulfate-6% polyacrylamide gelelectrophoresis, followed by silver staining. CIF130 can be separatedfrom CIF150/hTAF150 by excising the CIF130-containing band from the SDSgel and eluting the CIF130 protein, as is known in the art. Apreparation of isolated and purified CIF130 protein is at least 80%pure; preferably, the preparations are 90%, 95%, or 99% pure.

[0036] CIF130 proteins and polypeptides can also be produced byrecombinant DNA methods or by synthetic chemical methods. For productionof recombinant CIF130 proteins or polypeptides, coding sequencesselected from the CIF130 nucleotide sequence shown in SEQ ID NO: 1 canbe expressed in known prokaryotic or eukaryotic expression systems (seebelow). To avoid non-specific T7 RNA polymerase transcription infunctional assays, in vitro-translated CIF130 can be purified withNi-NTA-agarose as described above (100 mM imidazole eluate) andconcentrated with a Centricon 30 concentrator (Amicon). Bacterial,yeast, insect, or mammalian expression systems can be used, as is knownin the art. Alternatively, synthetic chemical methods, such as solidphase peptide synthesis, can be used to synthesize CIF130 protein orpolypeptides. CIF130 variants can be similarly produced.

[0037] Fusion proteins comprising at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 or more contiguous CIF130 amino acids canalso be constructed. Human CIF130 fusion proteins are useful forgenerating antibodies against CIF130 amino acid sequences and for use invarious assay systems. For example, CIF130 fusion proteins can be usedto identify proteins which interact with CIF130 protein and influenceits function. Physical methods, such as protein affinity chromatography,or library-based assays for protein-protein interactions, such as theyeast two-hybrid or phage display systems, can also be used for thispurpose. Such methods are well known in the art and can also be used asdrug screens, as described below

[0038] A CIF130 fusion protein comprises two protein segments fusedtogether by means of a peptide bond The first protein segment comprisesat least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, 30, 35, 40,50, 75, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or morecontiguous amino acids of a CIF130 protein. The amino acids can beselected from the amino acid sequence shown in SEQ ID NO: 2 or from abiologically active variant of that sequence, such as those describedabove. The first protein segment can also comprise a full-length CIF130protein as shown in SEQ ID NO: 2 or an alternate form of CIF130 as shownin FIG. 1. The first protein segment can be N-terminal or C-terminal, asis convenient.

[0039] The second protein segment can be a full-length protein or aprotein fragment or polypeptide. Proteins commonly used in fusionprotein construction include β-galactosidase, β-glucuronidase, greenfluorescent protein (GFP), autofluorescent proteins, including bluefluorescent protein (BFP), glutathione-S-transferase (GST), luciferase,horseradish peroxidase (HRP), and chloramphenicol acetyltransferase(CAT). Epitope tags can be used in fusion protein constructions,including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA)tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusionconstructions can include maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions.

[0040] CIF130 fusion proteins can be made by covalently linking thefirst and second protein segments or by standard procedures in the artof molecular biology. Recombinant DNA methods can be used to prepareCIF130 fusion proteins, for example, by making a DNA construct whichcomprises coding sequences selected from SEQ ID NO: 1 in proper readingframe with nucleotides encoding the second protein segment andexpressing the DNA construct in a host cell, as is known in the art.Many kits for constructing fusion proteins are available from companieswhich supply research labs with tools for experiments, including, forexample, Promega Corporation (Madison, Wis.), Stratagene (La Jolla,Calif.). Clontech (Mountain View, Calif.), Santa Cruz Biotechnology(Santa Cruz. Calif.), MBL International Corporation (MIC; Watertown,Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[0041] Isolated and purified CIF130 proteins, polypeptides, variants, orfusion proteins can be used as immunogens, to obtain a preparation ofantibodies which specifically bind to a CIF130 protein. The antibodiescan be used, inter alia, to detect wild-type CIF130 proteins in humantissue and fractions thereof The antibodies can also be used to detectthe presence of mutations in the CIF130 gene which result in under- oroverexpression of the CIF130 protein or in expression of a CIF130protein with altered size or electrophoretic mobility.

[0042] Antibodies which specifically bind to epitopes of CIF130proteins, polypeptides, fusion proteins, or biologically active variantscan be used in immunochemical assays, including but not limited toWestern blots, ELISAs, radioimmunoassays, immunohistochemical assays,immunoprecipitations, or other immunochemical assays known in the art.Typically, antibodies of the invention provide a detection signal atleast 5-, 10-, or 20-fold higher than a detection signal provided withother proteins when used in such immunochemical assays. Preferably,antibodies which specifically bind to CIF130 epitopes do not detectother proteins in immunochemical assays and can immunoprecipitate CIF130protein or polypeptides from solution.

[0043] CIF130-specific antibodies specifically bind to epitopes presentin a CIF130 protein having the amino acid sequence shown in SEQ ID NO: 2or to biologically active variants of that sequence. Typically, at least6, 8, 10, or 12 contiguous amino acids are required to form an epitope.However, epitopes which involve non-contiguous amino acids may requiremore, e.g., at least 15, 25, or 50 amino acids. Preferably, CIF130epitopes are not present in other human proteins.

[0044] Epitopes of CIF130 which are particularly antigenic can beselected, for example, by routine screening of CIF130 polypeptides forantigenicity or by applying a theoretical method for selecting antigenicregions of a protein to the amino acid sequence shown in SEQ ID NO: 2.Such methods are taught, for example, in Hopp and Wood, Proc. Natl.Acad. Sci. U.S.A. 78, 3824-28 (1981), Hopp and Wood, Mol. Immunol. 20,483-89 (1983), and Sutcliffe et al., Science 219, 660-66 (1983).

[0045] Any type of antibody known in the art can be generated to bindspecifically to CIF130 epitopes For example, preparations of polyclonaland monoclonal antibodies can be made using standard methods which arewell known in the art. Similarly, single-chain antibodies can also beprepared. Single-chain antibodies which specifically bind to CIF130epitopes can be isolated, for example, from single-chain immunoglobulindisplay libraries, as is known in the art. The library is “panned”against CIF130 amino acid sequences, and a number of single chainantibodies which bind with high-affinity to different epitopes of CIF130protein can be isolated. Hayashi et al., 1995, Gene 160-129-30.Single-chain antibodies can also be constructed using a DNAamplification method, such as the polymerase chain reaction (PCR), usinghybridoma cDNA as a template Thirion et al., 1996, Eur. J. Cancer Prev.5:507-11.

[0046] Single-chain antibodies can be mono- or bispecific, and can bebivalent or tetravalent. Construction of tetravalent, bispecificsingle-chain antibodies is taught, for example, in Coloma and Morrison,1997, Nat. Biotechnol. 15:159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught inter alia in Mallender and Voss,1994, J Biol. Chem. 269:199-206.

[0047] A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology. Verhaar et al., 1995,Int. J. Cancer 61:497-501, Nicholls et al., 1993, J. Immunol. Meth.165:81-91.

[0048] Monoclonal and other antibodies can also be “humanized” in orderto prevent a patient from mounting an immune response against theantibody when it is used therapeutically. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or may require alteration of a few key residues Sequencedifferences between, for example, rodent antibodies and human sequencescan be minimized by replacing residues which differ from those in thehuman sequences, for example, by site directed mutagenesis of individualresidues, or by grafting of entire complementarity determining regions.Alternatively, one can produce humanized antibodies using recombinantmethods, as described in GB2188638B. Antibodies which specifically bindto CIF130 epitopes can contain antigen binding sites which are eitherpartially or fully humanized, as disclosed in U S. Pat. No. 5,565,332.

[0049] Other types of antibodies can be constructed and used in methodsof the invention. For example, chimeric antibodies can be constructed asdisclosed, for example, in WO 93/03151. Binding proteins which arederived from immunoglobulins and which are multivalent andmultispecific, such as the “diabodies” described in WO 94/13804, canalso be prepared.

[0050] Antibodies of the invention can be purified by methods well knownin the art. For example, antibodies can be affinity purified by passingthe antibodies over a column to which a CIF130 protein, polypeptide,biologically active variant, or fusion protein is bound. The boundantibodies can then be eluted from the column, using a buffer with ahigh salt concentration.

[0051] CIF130-specific binding polypeptides other than antibodies canalso be generated. CIF130-specific binding polypeptides are polypeptideswhich bind with CIF130 or its variants and which have a measurablyhigher binding affinity for CIF130 and polypeptide derivatives of CIF130than for other polypeptides tested for binding. Higher affinity by afactor of 10 is preferred, more preferably a factor of 100. Suchpolypeptides can be found, for example, using the yeast two-hybridsystem.

[0052] SEQ ID NO: 1 represents a coding sequence of CIF130. Other formsof CIF130 are encoded by portions of SEQ ID NO: 1, utilizing any of thestart codons indicated in FIG. 1. The 38 basepair insertion indicated inFIG. 1 can be included or not, to form alternatively spliced forms ofCIF130 mRNA and protein. The complement of the nucleotide sequence shownin SEQ ID NO: 1 consists of a contiguous nucleotide sequence which formsWatson-Crick base pairs with the contiguous nucleotide sequences shownin SEQ ID NO: 1 and in FIG. 1.

[0053] Subgenomic CIF130 polynucleotides contain less than a wholechromosome Preferably, the polynucleotides are intron-free Purified andisolated CIF130 subgenomic polynucleotides can comprise at least 6, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 775, 780, 787, 788, 789, 800, 850, 900, 950, 1000, 1100, 1187,1188, 1189, 1190, 1191, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100,3200, 3300, 3350, 3351, 3352, 3400, 3500, 3600, 3700, or 3800 or morecontiguous nucleotides selected from SEQ ID NO: 1 or the codingsequences shown in FIG. 1 or their complements.

[0054] Antisense oligonucleotides, primers, and probes of the inventioncan consist of at least 11, 12, 15, 20, 25, 30, 50, or 100 contiguousnucleotides which are complementary to the coding sequences shown in SEQID NO: 1 and FIG. 1. A complement of the entire coding sequence can alsobe used Double-stranded subgenomic polynucleotides which comprise all ora portion of the nucleotide sequence shown in SEQ ID NO: 1, as well aspolynucleotides which encode CIF1 30-specific antibodies or ribozymes,are also subgenomic polynucleotides of the invention.

[0055] Degenerate nucleotide sequences encoding amino acid sequences ofCIF130 protein or biologically active CIF130 variants as well ashomologous nucleotide sequences which are at least 65%, 75%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown inSEQ ID NO: 1, are also CIF130 polynucleotides. Percent sequence identitybetween a polynucleotide having a nucleotide sequence selected from SEQID NO: 1 or the coding sequences shown in FIG. 1 and a putativehomologous or degenerate CIF130 nucleotide sequence is determined usingcomputer programs which employ the Smith-Waterman algorithm, for exampleas implemented in the MTSRCH program (Oxford Molecular), using an affinegap search with the following parameters: a gap open penalty of 12 and agap extension penalty of 1.

[0056] Nucleotide sequences which hybridize to a coding sequence shownin SEQ ID NO: 1 or FIG. 1 or their complements with at most 1, 2, 3, 4,5, 10, 15, 20, 25, 30, or 35% basepair mismatches are also CIF130subgenomic polynucleotides of the invention. For example, using thefollowing wash conditions—2×SSC (0 3 M sodium chloride, 0 03 M sodiumcitrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each,then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes, then 2×SSC, roomtemperature twice, 10 minutes each—homologous CIF130 sequences can beidentified which contain at most about 25-30% basepair mismatches with acoding sequence of SEQ ID NO: 1 or its complement. More preferably,homologous nucleic acid strands contain 15-25% basepair mismatches, evenmore preferably 5-15% basepair mismatches.

[0057] It is well known that the T_(m) of a double-stranded DNAdecreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al,J. Mol. Biol. 81, 123 (1973). Homologous CIF130 polynucleotides cantherefore be identified, for example, by hybridizing a putativehomologous CIF130 polynucleotide with a polynucleotide having a codingsequence selected from SEQ ID NO: 1 or the coding sequences shown inFIG. 1, comparing the melting temperature of the test hybrid with themelting temperature of a hybrid comprising a polynucleotide having thecoding sequence selected from SEQ ID NO: 1 or the coding sequences shownin FIG. 1 and a polynucleotide which is perfectly complementary to thecoding sequence, and calculating the number of basepair mismatcheswithin the test hybrid.

[0058] Nucleotide sequences which hybridize to coding sequences shown inSEQ ID NO: 1 or FIG. 1 or their complements following stringenthybridization and/or wash conditions are also CIF130 subgenomicpolynucleotides of the invention. Stringent wash conditions are wellknown and understood in the art and are disclosed, for example, inSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989,at pages 9.50-9.51.

[0059] Typically, for stringent hybridization conditions a combinationof temperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between the CIF130 sequence shown in SEQ IDNO: 1 and a polynucleotide sequence which is 65%, 75%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 can be calculated, forexample, using the equation of Bolton and McCarthy, Proc. Natl. Acad.Sci. U.S.A. 48, 1390 (1962):

T_(m)=81.5° C.−16 6(log₁₀[Na⁺])+0 41(% G+C)−0.63(% formamide)−600/l),where l=the length of the hybrid in basepairs.

[0060] Stringent wash conditions include, for example, 4×SSC at 65° C.,or 50% formamide, 4×SSC at 42° C., or 0 5×5X SSC, 0 1% SDS at 65° C.Highly stringent wash conditions include, for example, 0 2×SSC at 65° C.

[0061] CIF130 subgenomic polynucleotides can be isolated and purifiedfree from other nucleotide sequences using standard nucleic acidpurification techniques For example, restriction enzymes and probes canbe used to isolate subgenomic polynucleotide fragments which comprisethe CIF130 coding sequences Isolated and purified subgenomicpolynucleotides are in preparations which are free or at least 90% freeof other molecules.

[0062] Complementary DNA (cDNA) molecules which encode CIF130 proteinsare also CIF130 subgenomic polynucleotides of the invention. CIF130 cDNAmolecules can be made with standard molecular biology techniques, usingCIF130 mRNA as a template. CIF130 cDNA molecules can thereafter bereplicated using molecular biology techniques known in the art anddisclosed in manuals such as Sambrook et al, 1989. An amplificationtechnique, such as the polymerase chain reaction (PCR), can be used toobtain additional copies of subgenomic polynucleotides of the invention,using either human genomic DNA or cDNA as a template.

[0063] Alternatively, synthetic chemistry techniques can be used tosynthesize CIF130 subgenomic polynucleotide molecules of the invention.The degeneracy of the genetic code allows alternate nucleotide sequencesto be synthesized which will encode a CIF130 protein having the aminoacid sequence shown in SEQ ID NO: 2 or an alternate form of CIF130 asshown in FIG. 1 or a biologically active variant of one of thosesequences. All such nucleotide sequences are within the scope of thepresent invention.

[0064] The invention also provides polynucleotide probes which can beused to detect CIF130 sequences, for example, in hybridization protocolssuch as Northern or Southern blotting or in situ hybridizations.Polynucleotide probes of the invention comprise at least 12, 13, 14, 15,16, 17, 18, 19, 20, 30, or 40 or more contiguous nucleotides selectedfrom SEQ ID NO: 1 or a coding sequence shown in FIG. 1. Polynucleotideprobes of the invention can comprise a detectable label, such as aradioisotopic, fluorescent, enzymatic, or chemiluminescent label.

[0065] Purified and isolated CIF130 subgenomic polynucleotides can beused, inter alia, as primers to obtain additional copies of thepolynucleotides, to express human CIF130 mRNA, protein, polypeptides, orfusion proteins, as probes for identifying wild-type and mutant CIF130protein coding sequences, and to generate CIF130 antisenseoligonucleotides and ribozymes. For example, a CIF130 subgenomicpolynucleotide comprising CIF130 coding sequences can be used in anexpression construct, to express all or a portion of a CIF130 protein ina host cell. Host cells comprising CIF130 expression constructs can beprokaryotic or eukaryotic. Preferably, the CIF130 subgenomicpolynucleotide is inserted into an expression plasmid (for example, theEcdyson system, pIND, In Vitro Gene).

[0066] A variety of host cells are available for use in bacterial,yeast, insect, and human expression systems and can be used to expressCIF130 expression constructs (see below). Expression constructs of theinvention can be introduced into host cells using any technique known inthe art. These techniques include transferrin-polycation-mediated DNAtransfer, transfection with naked or encapsulated nucleic acids,liposome-mediated cellular fusion, intracellular transportation ofDNA-coated latex beads, protoplast fusion, viral infection,electroporation, and calcium phosphate-mediated transfection.

[0067] A CIF130 expression construct comprises a promoter which isfunctional in the particular host cell selected. The skilled artisan canreadily select an appropriate promoter from the large number of celltype-specific promoters known and used in the art. The expressionconstruct can also contain a transcription terminator which isfunctional in the host cell. The expression construct comprises apolynucleotide segment which encodes all or a portion of a CIF130protein, variant, fusion protein, antibody, or ribozyme. Thepolynucleotide segment is located downstream from the promoter.Transcription of the polynucleotide segment initiates at the promoter.The expression construct can be linear or circular and can containsequences, if desired, for autonomous replication

[0068] CIF130 subgenomic polynucleotides can be propagated in vectorsand cell lines using techniques well known in the art. CIF130 subgenomicpolynucleotides can be on linear or circular molecules. They can be onautonomously replicating molecules or on molecules without replicationsequences. They can be regulated by their own or by other regulatorysequences, as are known in the art

[0069] Bacterial systems for expressing CIF130 subgenomicpolynucleotides include those described in Chang et al., Nature (1978)275. 615, Goeddel et al., Nature (1979) 281: 544, Goeddel et al.,Nucleic Acids Res. (1980) 8 4057, EP 36,776, U.S. Pat. No. 4,551,433,deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25, andSiebenlist et al., Cell (1980) 20: 269.

[0070] Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al., J.Bacteriol. (1983) 153: 163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Gleeson et al.,J. Gen. Microbiol. (1986) 132: 3459, Roggenkamp et al., Mol. Gen. Genet.(1986) 202 :302) Das et al., J. Bacteriol. (1984) 158: 1165; DeLouvencourt et al., J. Bacteriol. (1983) 154: 737, Van den Berg et a.,Bio/Technology (1990) 8. 135; Kunze et al., J. Basic Microbiol. (1985)25: 141, Cregg et al., Mol. Cell. Biol. (1985).5 3376, U.S. Pat. No.4,837,148, U.S. Pat. No. 4,929,555; Beach and Nurse, Nature (1981) 300706; Davidow et al., Curr. Genet. (1985) 10: 380, Gaillardin et al.,Curr. Genet. (1985) 10: 49, Ballance et al., Biochem. Biophys. Res.Commun. (1983) 112: 284-289; Tilburn et al., Gene (1983) 26: 205-221,Yelton et al., Proc. Natl. Acad. Sci. USA (1984) 81: 1470-1474, Kellyand Hynes, EMBO J. (1985) 4: 475479; EP 244,234, and WO 91/00357.

[0071] Expression of CIF130 subgenomic polynucleotides in insects can becarried out as described in U.S. Pat. No. 4,745,051, Friesen et al(1986) “The Regulation of Baculovirus Gene Expression” in: THE MOLECULARBIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476, andVlak et al., J. Gen. Virol (1988) 69: 765-776, Miller et al., Ann. Rev.Microbiol (1988) 42: 177, Carbonell et al., Gene (1988) 73: 409, Maedaet al., Nature (1985) 315: 592-594, Lebacq-Verheyden et al., Mol. Cell.Biol. (1988) 8: 3129; Smith et al.,

[0072] Proc. Natl. Acad. Sci. USA (1985) 82: 8404, Miyajima et al., Gene(1987) 58: 273; and Martin et al., DNA (1988) 7:99. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts are described in Luckow et al, Bio/Technology (1988) 6: 47-55,Miller et al., in GENETIC ENGINEERING, (Setlow, J K. et al eds.), Vol. 8(Plenum Publishing, 1986), pp 277-279, and Maeda et al., Nature, (1985)315 592-594

[0073] Mammalian expression of CIF130 subgenomic polynucleotides can beachieved as described in Dijkema et al., EMBO J. (1985) 4-761, Gorman etal., Proc. Natl. Acad. Sci. USA (1982b) 79: 6777, Boshart et al., Cell(1985) 41: 521 and U.S. Pat. No. 4,399,216. Other features of mammalianexpression can be facilitated as described in Ham and Wallace, Meth.Enz. (1979) 58: 44, Barnes and Sato, Anal. Biochem. (1980) 102. 255, U.SPat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762,U.S. Pat. No. 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.

[0074] Polynucleotides of the invention can also be used in genedelivery vehicles, for the purpose of delivering a CIF130 mRNA oroligonucleotide (either with the sequence of native CIF130 mRNA or itscomplement), full-length CIF130 protein, CIF130 fusion protein, CIF130polypeptide, or CIF130-specific ribozyme or single-chain antibody, intoa cell preferably a eukaryotic cell. According to the present invention,a gene delivery vehicle can be, for example, naked plasmid DNA, a viralexpression vector comprising a CIF130 polynucleotide, or a CIF130polynucleotide in conjunction with a liposome or a condensing agent.

[0075] In one embodiment of the invention, the gene delivery vehiclecomprises a promoter and a CIF130 polynucleotide. Preferred promotersare tissue-specific promoters and promoters which are activated bycellular proliferation, such as the thymidine kinase and thymidylatesynthase promoters. Other preferred promoters include promoters whichare activatable by infection with a virus, such as the α- andβ-interferon promoters, and promoters which are activatable by ahormone, such as estrogen. Other promoters which can be used include theMoloney virus LTR, the CMV promoter, and the mouse albumin promoter.

[0076] A CIF30 gene delivery vehicle can comprise viral sequences suchas a viral origin of replication or packaging signal. These viralsequences can be selected from viruses such as astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, retrovirus, togavirus or adenovirus. In a preferredembodiment, the CIF130 gene delivery vehicle is a recombinant retroviralvector. Recombinant retroviruses and various uses thereof have beendescribed in numerous references including, for example, Mann et al.,Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA81.6349, 1984, Miller et al., Human Gene Therapy 15-14, 1990, U.S. Pat.Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral genedelivery vehicles can be utilized in the present invention, includingfor example those described in EP 0,415,731, WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234, U.S. Pat. No 5,219,740; WO 9311230; WO9310218, Vile and Hart, Cancer Res. 53:3860-3864, 1993, Vile and Hart,Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53.83-88, 1993;Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.Neurosurg. 79.729-735, 1993 (U.S Pat. No. 4,777,127, GB 2,200,651, EP0,345,242 and WO91/02805)

[0077] Packaging cell lines suitable for use with the above-describedretroviral gene delivery vehicles can be readily prepared (see Ser. No.08/240,030, filed May 9, 1994; see also WO 92/05266) and used to createproducer cell lines (also termed vector cell lines or “VCLs” ) forproduction of recombinant viral particles

[0078] A CIF130 gene delivery vehicle can also be a recombinantadenoviral gene delivery vehicle. Such vehicles can be readily preparedand utilized given the disclosure provided herein (see Berkner,Biotechniques 6:616, 1988, and Rosenfeld et al., Science 252:431, 1991,WO 93/07283, WO 93/06223, and WO 93/07282). Adeno-associated viralCIF130 gene delivery vehicles can also be constructed and used todeliver CIF130 amino acids or nucleotides. The use of adeno-associatedviral gene delivery vehicles in vitro is described in Chatterjee et al.,Science 258: 1485-1488 (1992), Walsh et al., Proc. Nat'l. Acad. Sci. 89:7257-7261 (1992), Walsh et al., J. Clin. Invest. 94: 1440-1448 (1994),Flotte et al., J. Biol. Chem. 268: 3781-3790 (1993), Ponnazhagan et al.,J. Exp. Med. 179: 733-738 (1994), Miller et al., Proc. Nat'l. Acad. Sci.91: 10183-10187 (1994), Einerhand et al., Gene Ther. 2: 336-343 (1995),Luo et al., Exp. Hematol. 23: 1261-1267 (1995), and Zhou et al., GeneTherapy 3: 223-229 (1996). In vivo use of these vehicles is described inFlotte et al., Proc. Nat'l Acad. Sci. 90: 10613-10617 (1993), andKaplitt et al., Nature Genet. 8:148-153 (1994).

[0079] In another embodiment of the invention, a CIF130 gene deliveryvehicle is derived from a togavirus. Preferred togaviruses includealphaviruses, in particular those described in U.S. Ser. No. 08/405,627,filed Mar. 15, 1995, WO 95/07994. Alpha viruses, including Sindbis andELVS viruses can be gene delivery vehicles for CIF130 polynucleotides.Alpha viruses are described in WO 94/21792, WO 92/10578 and WO 95/07994.Several different alphavirus gene delivery vehicle systems can beconstructed and used to deliver CIF130 polynucleotides to a cellaccording to the present invention. Representative examples of suchsystems include those described in U.S. Pat. Nos. 5,091,309 and5,217,879. Particularly preferred alphavirus gene delivery vehicles foruse in the present invention include those which are described in WO95/07994, and U.S. Ser. No. 08/405,627

[0080] A CIF130 polynucleotide of the invention can also be combinedwith a condensing agent to form a gene delivery vehicle In a preferredembodiment, the condensing agent is a polycation, such as polylysine,polyarginine, polyornithine, protamine, spermine, spermidine, andputrescine. Many suitable methods for making such linkages are known inthe art (see, for example, Ser. No. 08/366,787, filed Dec. 30, 1994).

[0081] In an alternative embodiment, a CIF130 polynucleotide isassociated with a liposome to form a gene delivery vehicle. See Stryer,Biochemistry, pp. 236-240, 1975 (W. H. Freeman, San Francisco, Calif.);Szoka et al., Biochim. Biophys. Acta 600:1, 1980; Bayer et al., Biochim.Biophys. Acta. 550:464, 1979; Rivnay et al., Meth. Enzymol. 149:119,1987; Wang et al., Proc. Natl. Acad Sci. U.S.A. 84: 7851, 1987, Plant etal., Anal. Biochem. 176:420, 1989, and U.S. Pat. No. 4,762,915.Liposomes can encapsulate a variety of nucleic acid molecules includingDNA, RNA, plasmids, and expression constructs comprising CIF130polynucleotides such those disclosed in the present invention.

[0082] In addition, lipoproteins can be included with a CIF130polynucleotide for delivery to a cell. Examples of such lipoproteinsinclude chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, orfusions of these proteins can also be used. Modifications of naturallyoccurring lipoproteins can also be used, such as acetylated LDL. Theselipoproteins can target the delivery of polynucleotides to cellsexpressing lipoprotein receptors. Preferably, if lipoproteins areincluded with a polynucleotide, no other targeting ligand is included inthe composition.

[0083] In another embodiment, naked CIF130 polynucleotide molecules areused as gene delivery vehicles, as described in WO 90/11092 and U.S.Pat. No. 5,580,859 Such gene delivery vehicles can be either CIF130 DNAor RNA and, in certain embodiments, are linked to killed adenovirus.Curiel et al., Hum. Gene. Ther. 3-147-154, 1992. Other suitable vehiclesinclude DNA-ligand (Wu et al., J. Biol. Chem. 264 16985-16987, 1989),lipid-DNA combinations (Felgner et al., Proc. Natl. Acad. Sci. USA 847413 7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci.84:7851-7855, 1987) and microprojectiles (Williams et al., Proc. Nail.Acad. Sci. 88:2726-2730, 1991)

[0084] The invention provides compositions for regulating cell cycleprogression, in order to alter spatial or temporal patterns of divisionof a human cell. CIF130 binds to CIF150/hTAF_(II)150 and inhibits cellcycle progression, indicating that CIF130 negatively regulatesCIF150/hTAF_(II)150 activity. CIF150/hTAF_(II)150 is required for a cellto enter mitosis. Thus, mitosis or cell cycle progression can be inducedor increased by increasing expression of a human CIF130 gene. IncreasedCIF130 gene expression can be used to expand cell populations in vitroor for treating disorders such as anemia, which are characterized bylowered rates of mitosis. Cells in which expression of a CIF130 gene hasbeen decreased can also be used to identify genes whose expression isdependent on a CIF 130 protein. Decreased CIF130 gene expression can beused to treat conditions characterized by high rates of mitosis, such asneoplasia, metastasis of neoplasms, benign proliferative diseases, anddysplastic and hyperplastic disorders.

[0085] In one embodiment of the invention, expression of a CIF130 geneis decreased using a ribozyme, an RNA molecule with catalytic activity.See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, 1990, Ann. Rev.Biochem. 59:543-568; Cech, 1992, Curr. Opin. Struct. Biol. 2: 605-609;Couture and Stinchcomb, 1996, Trends Genet. 12-510-515 Ribozymes can beused to inhibit gene function by cleaving an RNA sequence, as is knownin the art (e.g., Haseloff et al., U.S. Pat. No. 5,641,673).

[0086] The coding sequence of the CIF130 gene can be used to generateribozymes which will specifically bind to mRNA transcribed from a CIF130gene. Methods of designing and constructing ribozymes which can cleaveother RNA molecules in trans in a highly sequence specific manner havebeen developed and described in the art (see Haseloff et al. (1988),Nature 334-585-591). For example, the cleavage activity of ribozymes canbe targeted to specific CIF130 RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target CIF130 RNA and thusspecifically hybridizes with the target (see, for example, Gerlach etal., EP 321,201). The nucleotide sequence shown in SEQ ID NO: 1 providesa source of suitable hybridization region sequences. Longercomplementary sequences can be used to increase the affinity of thehybridization sequence for the target. The hybridizing and cleavageregions of the CIF130 ribozyme can be integrally related; thus, uponhybridizing to the target CIF130 RNA through the complementary regions,the catalytic region of the ribozyme can cleave the target.

[0087] CIF130 ribozymes can be introduced into cells as part of a DNAconstruct, as is known in the art and described above. Mechanicalmethods, such as microinjection, liposome-mediated transfection,electroporation, or calcium phosphate precipitation, can be used tointroduce the ribozyme-containing DNA construct into cells in which itis desired to decrease CIF130 expression, as described above.Alternatively, if it is desired that the cells stably retain the DNAconstruct, it can be supplied on a plasmid and maintained as a separateelement or integrated into the genome of the cells, as is known in theart. The DNA construct can include transcriptional regulatory elements,such as a promoter element, an enhancer or UAS element, and atranscriptional terminator signal, for controlling transcription ofCIF130 ribozymes in the cells.

[0088] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, CIF130ribozymes can be engineered so that ribozyme expression will occur inresponse to factors which induce expression of the CIF130 gene.Ribozymes can also be engineered to provide an additional level ofregulation, so that destruction of CIF130 mRNA occurs only when both aCIF130 ribozyme and a CIF130 gene are induced in the cells.

[0089] In another embodiment of the invention, expression of the CIF130gene is altered using an antisense oligonucleotide sequence. Theantisense sequence is complementary to at least a portion of the codingsequence of a CIF130 gene having the nucleotide sequence shown in SEQ IDNO: 1 Preferably, the antisense oligonucleotide sequence is at least sixnucleotides in length, but can be about 8, 9, 10, 11, 12, 15, 20, 25,30, 35, 40, 45, or 50 nucleotides long. Longer sequences can also beused. CIF130 antisense oligonucleotide molecules can be provided in aDNA construct and introduced into cells as described above to decreaseCIF130 expression.

[0090] CIF130 antisense oligonucleotides can be deoxyribonucleotides,ribonucleotides, or a combination of both Oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with non-phosphodiester internucleotide linkages such asalkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux,1994, Meth. Mol. Biol. 26: 1-72; Uhlmann et al., 1990, Chem. Rev.90:543-583.

[0091] Precise complementarity is not required for successful duplexformation between an antisense molecule and the complementary codingsequence of a CIF130 gene. Antisense molecules which comprise, forexample, 2, 3, 4, or 5 or more stretches of contiguous nucleotides whichare precisely complementary to a CIF130 coding sequence, each separatedby a stretch of contiguous nucleotides which are not complementary toadjacent CIF130 coding sequences, can provide targeting specificity forCIF130 mRNA. Preferably, each stretch of contiguous nucleotides is atleast 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementaryintervening sequences are preferably 1, 2, 3, or 4 nucleotides inlength. One skilled in the art can easily use the calculated meltingpoint of an antisense-sense pair to determine the degree of mismatchingwhich will be tolerated between a particular antisense oligonucleotideand a particular CIF130 coding sequence.

[0092] CIF130 antisense oligonucleotides can be modified withoutaffecting their ability to hybridize to a CIF130 coding sequence Thesemodifications can be internal or at one or both ends of the antisensemolecule. For example, internucleoside phosphate linkages can bemodified by adding cholesteryl or diamine moieties with varying numbersof carbon residues between the amino groups and terminal ribose.Modified bases and/or sugars, such as arabinose instead of ribose, or a3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the5′ phosphate group are substituted, can also be employed in a modifiedantisense oligonucleotide These modified oligonucleotides can beprepared by methods well known in the art. See, e.g., Agrawal et al,1992, Trends Biotechnol. 10 152-158, Uhlmann et al, 1990, Chem. Rev.90-543-584; Uhlmann et al, 1987, Tetrahedron. Lett. 215 3539-3542.

[0093] Antibodies of the invention which specifically bind to a CIF130protein, particularly single-chain antibodies, can also be used to alterCIF130 gene expression. CIF130-specific antibodies bind to CIF130protein and prevent the protein from functioning in the cell.Polynucleotides encoding single-chain antibodies of the invention can beintroduced into cells as described above.

[0094] Preferably, the mechanism used to decrease expression of theCIF130 gene, whether ribozyme, antisense nucleotide sequence, orantibody, decreases expression of the CIF130 gene by 50%, 60%, 70%, or80%. Most preferably, expression of the CIF130 gene is decreased by 90%,95%, 99%, or 100%. The effectiveness of the mechanism chosen to alterexpression of the CIF130 gene can be assessed using methods well knownin the art, such as hybridization of nucleotide probes to CIF130 mRNA,quantitative RT-PCR, or detection of CIF130 protein usingCIF130-specific antibodies of the invention.

[0095] Compositions comprising CIF130 antibodies, ribozymes, orantisense oligonucleotides can be used to increase the number of cellsin a cell population in vitro or for treating disorders characterized bylowered rates of mitosis, such as anemia. CIF130 compositions of theinvention can optionally comprise a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known to those in the art.Such carriers include, but are not limited to, large, slowly metabolizedmacromolecules, such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andinactive virus particles. Pharmaceutically acceptable salts can also beused in CIF130 compositions, for example, mineral salts such ashydrochlorides, hydrobromides, phosphates or sulfates, as well as saltsof organic acids such as acetates, proprionates, malonates, orbenzoates. CIF130 compositions can also contain liquids, such as water,saline, glycerol, and ethanol, as well as substances such as wettingagents, emulsifying agents, or pH buffering agents. Liposomes, such asthose described in U.S. Pat. No. 5,422,120, WO 95/13796, WO 91/14445, orEP 524,968 B 1, can also be used as a carrier for a CIF130 composition.

[0096] Typically, a CIF130 composition is prepared as an injectable,either as a liquid solution or suspension; however, solid forms suitablefor solution or suspension in liquid vehicles prior to injection canalso be prepared A CIF130 composition can also be formulated into anenteric coated tablet or gel capsule according to known methods in theart, such as those described in U.S Pat. No. 4,853,230, EP 225,189, AU9,224,296, and AU 9,230,801.

[0097] Alternatively, a composition comprising all or a portion of aCIF130 gene or expression product can be introduced into a cell in orderto suppress or decrease mitosis or cell cycle progression. Suchcompositions can also comprise a pharmaceutically acceptable carrier, asdescribed above. Proliferative disorders, such as neoplasias,dysplasias, and hyperplasias, and their symptoms can be treated byadministration of a CIF130 composition comprising coding sequences forCIF130 or comprising CIF130 proteins or protein fragments. Neoplasiaswhich can be treated with such CIF130 compositions include, but are notlimited to, melanomas, squamous cell carcinomas, adenocarcinomas,hepatocellular carcinomas, renal cell carcinomas, sarcomas, myosarcomas,non-small cell lung carcinomas, leukemias, lymphomas, osteosarcomas,central nervous system tumors such as gliomas, astrocytomas,oligodendrogliomas, and neuroblastomas, tumors of mixed origin, such asWilms' tumor and teratocarcinomas, and metastatic tumors. Proliferativedisorders which can be treated with a CIF130 composition comprisingCIF130 coding sequences or CIF130 proteins or polypeptides includedisorders such as anhydric hereditary ectodermal dysplasia, congenitalalveolar dysplasia, epithelial dysplasia of the cervix, fibrousdysplasia of bone, and mammary dysplasia. Hyperplasias, for example,endometrial, adrenal, breast, prostate, or thyroid hyperplasias, orpseudoepitheliomatous hyperplasia of the skin can be treated with suchCIF130 compositions.

[0098] An entire CIF130 coding sequence or protein can be introduced, asdescribed above. Alternatively, a portion of a CIF130 protein whichbinds to CIF150/hTAF_(II)150 and inhibits mitosis or cell cycleprogression can be identified and that portion or a nucleotide sequenceencoding it can be introduced into the cell Portions of a CIF130 proteinwhich result in decreased mitosis or cell cycle progression can beidentified by introducing expression constructs which express differentportions of the protein into cells and measuring alterations in the rateof mitosis. Rates of mitosis can be measured, for example, by detectingincorporation of labeled nucleotides, as is known in the art.

[0099] Even in disorders in which CIF130 mutations are not implicated,increasing CIF130 function can have therapeutic application In thesedisorders, increasing CIF130 expression or function can help to suppresstumors. Similarly, in tumors in which CIF130 expression is not aberrant,CIF130 upregulation or increase of CIF130 activity can help to suppressmetastases.

[0100] CIF130 compositions which contain CIF130 subgenomicpolynucleotides preferably contain an expression construct comprising apromoter and a polynucleotide segment encoding at least 17 contiguousamino acids of a CIF130 protein or protein variant. Within theexpression construct, the polynucleotide segment is located downstreamfrom the promoter, and transcription of the polynucleotide segmentinitiates at the promoter. A more complete description of gene transfervectors, especially retroviral vectors, is contained in U.S. Ser. No.08/869,309.

[0101] Administration of CIF130 compositions of the invention caninclude local or systemic administration, including injection, oraladministration, particle gun, or catheterized administration, andtopical administration. Various methods can be used to administer aCIF130 composition directly to a specific site in the body. For example,a small metastatic lesion can be located and an appropriate CIF130composition injected several times in several different locations withinthe body of the lesion. Alternatively, arteries which serve a tumor canbe identified, and a CIF130 composition can be injected into such anartery in order to deliver the composition to the tumor.

[0102] A tumor which has a necrotic center can be aspirated, and aCIF130 composition can be injected directly into the now empty center ofthe tumor. A CIF130 composition can also be administered directly to thesurface of a tumor, for example, by topical application of thecomposition. X-ray imaging can be used to assist in certain of thesedelivery methods. Combination therapeutic agents, including a CIF130protein, polypeptide, or subgenomic CIF130 polynucleotide together withother therapeutic agents, can be administered simultaneously orsequentially.

[0103] CIF130 compositions can be delivered to specific tissues usingreceptor-mediated targeted delivery. Receptor-mediated DNA deliverytechniques are taught in, for example, Findeis et al. (1993), Trends inBiotechnol. 11, 202-05, Chiou et al (1994), GENE THERAPEUTICS: METHODSAND APPLICATIONS OF DIRECT GENE TRANSFER (J A Wolff, ed.); Wu & Wu(1988), i J. Biol. Chem. 263, 621-24; Wu et al (1994), J. Biol. Chem.269, 542-46; Zenke et al. (1990), Proc. Natl. Acad Sci. U.S.A. 87,3655-59; Wu et al. (1991), J. Biol. Chem. 266, 338-42

[0104] Alternatively, a CIF130 composition can be introduced into humancells ex vivo, and the cells then replaced into the human. Cells can beremoved from a variety of locations including, for example, from aselected tumor or from an affected organ. A CIF130 composition can beinserted into non-tumorigenic cells, such as dermal fibroblasts orperipheral blood leukocytes. If desired, particular fractions of cells,such as T cell subsets or stem cells, can also be specifically removedfrom the blood (see, for example, PCT WO 91/16116). The removed cellscan then be contacted with a CIF130 composition utilizing any of theabove-described techniques, followed by the return of the cells to thehuman.

[0105] Both the dose of a particular CIF130 composition and the means ofadministering the composition can be determined based on specificqualities of the CIF130 composition, the condition, age, and weight ofthe patient, the progression of the particular disease being treated,and other relevant factors. If the composition contains CIF130 proteins,polypeptides, or antibodies, effective dosages of the composition are inthe range of about 5 fig to about 50 μg/kg of patient body weight, about50 μg to about 5 μg/kg, about 100 μg to about 500 μg/kg of patient bodyweight, and about 200 to about 250 μg/kg.

[0106] Compositions containing CIF130 subgenomic polynucleotides,including antisense oligonucleotides and ribozyme-or antibody-encodingsequences, can be administered in a range of about 100 ng to about 200mg of DNA for local administration. Suitable concentrations range fromabout 500 ng to about 50 μg, about 1 μg to about 2 mg, about 5 μg toabout 500 μg, and about 20 μg to about 100 μg of DNA. Factors such asmethod of action and efficacy of transformation and expression areconsiderations which will affect the dosage required for ultimateefficacy of the CIF130 composition. If greater expression is desiredover a larger area of tissue, larger amounts of CIF130 compositions orthe same amount administered successively, or several administrations todifferent adjacent or close tissue portions of, for example, a tumorsite, may be required to effect a positive therapeutic outcome. In allcases, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect.

[0107] Expression of an endogenous CIF130 gene in a cell can be alteredby introducing in frame with the endogenous CIF130 gene a DNA constructcomprising a CIF130 targeting sequence, a regulatory sequence, an exon,and an unpaired splice donor site by homologous recombination, such thata homologously recombinant cell comprising a new CIF130 transcriptionunit is formed. The new transcription unit can be used to turn theCIF130 gene on or off as desired. This method of affecting endogenousgene expression is taught in U.S. Pat. No. 5,641,670, which isincorporated herein by reference.

[0108] The targeting sequence is a segment of at least 10, 12, 15, 20,or 50 contiguous nucleotides selected from the nucleotide sequence shownin SEQ ID NO: 1. The transcription unit is located upstream of a codingsequence of the endogenous CIF130 gene. The exogenous regulatorysequence directs transcription of the coding sequence of the CIF130gene.

[0109] The present invention also provides a method of diagnosing orprognosing neoplasia or of identifying neoplastic tissue of a human.CIF130 mRNA is apparently overexpressed in tumors when total RNA iscompared (see Example 2). Expression of a CIF130 gene can therefore becompared between a first tissue which is suspected of being neoplasticand a second tissue of the human which is normal. The first and secondtissues can be obtained from the same human or from different humans.The normal tissue can be any tissue of the human, including, but notlimited to, spleen, thymus, prostate, testis, ovary, small intestine,mucosal lining of the colon, peripheral blood leukocytes, heart, glialcells, placenta, lung, liver, skeletal muscle, kidney, pancreas,peripheral blood leukocytes, bone marrow, and appendix. The tissuesuspected of being neoplastic can be derived from a different tissuetype of the human, but preferably it is derived from the same tissuetype, for example an intestinal polyp or other abnormal growth.

[0110] Overexpression of the CIF130 gene in the suspect tissueidentifies the suspect tissue as neoplastic. Expression of CIF130 can bedetected by measuring total CIF130 mRNA. Total RNA can be isolated fromthe two tissues, as is known in the art. One of skill in the art canreadily determine differences in the size or amount of CIF130 mRNAtranscripts between the two tissues that are compared, using Northernblots or in situ hybridization with nucleotide probes selected from thenucleotide sequence shown in SEQ ID NO: 1. Overexpression of CIF130 mRNAin a tissue sample suspected of being neoplastic compared with theexpression of CIF130 mRNA in a normal tissue is indicative of neoplasia.

[0111] Alternatively, CIF130 proteins can be compared between the twosamples. Any method for analyzing proteins can be used to compare twoCIF130 proteins from matched samples. The sizes of the CIF130 proteinsin the two tissues can be compared, for example, using antibodies of theinvention to detect CIF130 proteins in Western blots of protein extractsfrom the two tissues. Other changes, such as expression levels andsubcellular localization, can also be detected immunologically. A higherCIF130 protein expression level in a tissue suspected of beingneoplastic compared with the CIF130 protein expression level in a normaltissue is indicative of neoplasia.

[0112] Similarly, comparison of CIF130 gene sequences or of CIF130 geneexpression products, e.g., mRNA and protein, between a tissue of a humanwhich is suspected of being neoplastic and a normal tissue of a humancan be used to diagnose or prognose neoplasia in the human. The CIF130genes in the two tissues can be compared by any means known in the art.For example, the two genes can be sequenced, and the sequence of theCIF130 gene in the tissue suspected of being neoplastic, can be comparedwith the wild-type CIF130 sequence in the normal tissue. The CIF130genes or portions of the CIF130 genes in the two tissues can beamplified, for example, using nucleotide primers selected from thenucleotide sequence shown in SEQ ID NO: 1 in the polymerase chainreaction (PCR) or other amplification technique. The amplified genes orportions of genes can be hybridized to nucleotide probes selected fromthe nucleotide sequence shown in SEQ ID NO: 1 The nucleotide probes canbe labeled by a variety of methods, such as radiolabeling,biotinylation, or coupling to fluorescent or chemiluminescent tags, anddetected by standard methods known in the art. Comparisons of CIF130mRNA or protein can be made as described above. A difference between theCIF130 gene (or a gene which regulates, for example, its expression,half-life, or degradation) in the two tissues which are comparedindicates neoplasia in the tissue The degree of overexpression of theCIF130 gene in the neoplastic tissue relative to wild-type expression ofthe gene in normal tissue, or differences in the amount ofoverexpression of the CIF130 gene in the neoplastic tissue over time,can be used to prognose the progression of the neoplasia in that tissueor to monitor the response of the neoplastic tissue to varioustherapeutic regimens.

[0113] A genetic predisposition to neoplasia in a human can be detectedby comparing a wild-type CIF130 gene, mRNA, or protein with a CIF130gene, mRNA, or protein in a fetal tissue. Fetal tissues which can beused for this purpose include, but are not limited to, amniotic fluid,chorionic villi, blood, and the blastomere of an in vitro-fertilizedembryo. The wild-type CIF130 gene can be obtained from any tissue. ThemRNA or protein can be obtained from a normal tissue of a human in whichthe CIF130 gene is expressed. Such tissues are disclosed above.Differences, such as alterations in the nucleotide sequence or size ofthe fetal CIF130 gene or mRNA, or alterations in the molecular weight,amino acid sequence, or relative abundance of fetal CIF130 protein,indicate a germline mutation in the CIF130 gene of the fetus whichindicates a genetic predisposition to neoplasia.

[0114] Kits for use in the diagnostic methods described above are alsoprovided. CIF130 diagnostic kits comprise reagents which specificallybind to a human CIF130 gene or expression product and which can be usedin methods of the invention, such as CIF130 subgenomic polynucleotideprobes or antibodies. Means for labeling the probes or antibodies,reagents for use in the methods, such as buffers, and instructions forusing the kits can also be included.

[0115] The function of CIF130 as a negative regulator of thetranscription factor CIF150/hTAF_(II)150 can be exploited to identifygenes whose transcription is dependent on the presence of CIF130.Isolated RNA from the two populations can be compared to identify geneswhich are differentially transcribed in two cell populations. In onepopulation of a cell type, such as HeLa or NIH 3T3 cells, expression ofa CIF130 gene is unaltered; in the other population of the cell type,expression of a CIF130 gene is decreased. RNA can be isolated from thetwo populations by methods well known in the art.

[0116] Decreased CIF130 expression can be achieved, for example, usingribozymes, antisense oligonucleotide sequences, or antibodies, asdescribed above The effectiveness of the mechanism chosen to alterexpression of the CIF130 gene can be assessed using methods well knownin the art, such as hybridization of nucleotide probes to CIF130 mRNA ordetection of CIF130 protein using specific antibodies Genes which aredifferentially transcribed in the two populations can be compared, forexample, using differential display PCR. Differential display PCR can becarried out on the two populations of cells using methods well known inthe art. See, e.g., Liang & Pardee, 1992, Science 257:967-71, Bauer etal, 1993, Nucl. Acids. Res. 21:4272-80; Bauer et al., 1994, PCR MethodsAppl. 4:S97-108; and Liang et al., 1995, Meth. Enz. 254:30421. Kits forperforming differential display PCR are available, for example, fromDisplay Systems Biotech.

[0117] Briefly, total RNA is isolated form the two populations of cells.The RNA is reverse transcribed to produce a cDNA population whichrepresents an overlapping subset of the total expression profile of thecells in each population. Each subset cDNA population is amplified usingPCR with an anchored primer and a group of arbitrary primers in thepresence of radiolabeled dATP. Amplified products from the twopopulations of cells are separated by gel electrophoresis, and patternsof separated products are detected, as is known in the art.

[0118] Differences in the two patterns, such as the presence, absence,altered position within the gel, or amount of one or more cDNA species,indicates that the expression of one or more genes was altered inresponse to decreasing the expression of the CIF130 gene. Differentiallydisplayed bands can be excised from the gel, reamplified, and identifiedby sequence analysis. Optionally, the sequences can be cloned beforesequencing. Sequences of the differentially displayed bands can becompared with known sequences in databases to determine the identity ofgenes whose expression was altered in response to decreasing expressionof CIF130.

[0119] The invention also provides means of identifying compounds whichalter mitosis or cell cycle progression. A cell population is contactedwith a test compound. A test compound can be a pharmacologic agentalready known in the art or can be a compound previously unknown to haveany pharmacological activity. A test compound can be naturally occurringor designed in the laboratory. It can be isolated from microorganisms,animals, or plants, and can be produced recombinantly, or synthesized bychemical methods known in the art.

[0120] The cell population can comprise any primary human cell or humancell line which expresses a CIF130 gene, as disclosed above. Methods ofestablishing cultures of primary human cells or of culturing cell linesare well known in the art.

[0121] Expression of the CIF130 gene in the cell population is detected.Means of detecting CIF130 gene expression, for example by measuringCIF130 mRNA or CIF130 protein, are disclosed above. Expression can bemeasured in a sample of the same cell population before and aftercontact with the test compound. Alternatively, control cell populationswhich have not been contacted with the test compound can be employed. Atest compound which decreases expression of the CIF130 gene isidentified as a potential compound for inducing mitosis or cell cycleprogression. A test compound which increases expression of the CIF130gene is identified as a potential compound for inhibiting mitosis orcell cycle progression.

[0122] The invention also provides methods for screening test compoundsfor the ability to interfere with the binding of CIF130 toCIF150/hTAF_(II)150 According to one method, a CIF130 and aCIF150/hTAF_(II)150 protein, or at least the domains of each proteinnecessary for the binding interaction, are incubated together in thepresence of a test compound. In the absence of the test compound, theCIF150/hTAF_(II)150-binding domain of the CIF130 protein binds to theCIF130-binding domain of CIF150/hTAF_(II)150 The amount of bound and/orunbound proteins or binding domains is determined according to anytechnique known in the art, including any immunological technique Inorder to facilitate the assay, one of the proteins or binding domainscan be bound to a solid support, or can be labeled with a radiolabel, orother detectable label. A useful agent is identified which decreases theamount of CIF130 and/or CIF150/hTAF_(II)150 protein or binding domainwhich is bound or increases the amount of CIF130 and/orCIF150/hTAF_(II)150 protein or binding domain which is unbound. TheCIF130 and CIF150/hTAF_(II)150 proteins or binding domains can beprebound prior to the introduction of the test compound, or the testcompound can be contacted with one of the two proteins or bindingdomains prior to incubation.

[0123] In another embodiment, a two-hybrid assay is used to screencompounds which inhibit the interaction between the binding partners,CIF130 and CIF150/hTAF_(II)150 According to such an assay, a fusionprotein of each of the binding partners is used. The fusion proteins cancomprise full-length CIF130 or CIF150/hTAF_(II)150 proteins or at leastthe domains of each protein necessary for the binding interaction. Oneof the binding partners is fused to a DNA binding domain and the otheris fused to a transcriptional activating domain. If the fusion proteincomprising the CIF130 protein or CIF150/hTAF_(II)150-binding domain ofthe CIF130 protein comprises the transcriptional activating domain, thenthe fusion protein comprising the CIF150/hTAF_(II)150 protein orCIF130-binding domain of the CIF150/hTAF_(II)50 protein comprises theDNA binding domain. If the fusion protein comprising theCIF150/hTAF_(II)150 protein or CIF130-binding domain of theCIF150/hTAF_(II)150 protein comprises the transcriptional activatingdomain, then the fusion protein comprising the CIF130 protein orCIF150/hTAF_(II)150-binding domain of the CIF130 protein comprises theDNA binding domain. The two fusion proteins interact to reconstitute asequence-specific transcriptional activating factor. Many DNA bindingdomains and transcriptional activating domains can be used in thissystem, including the DNA binding domains of GAL4, LexA, and the humanestrogen receptor paired with the acidic transcriptional activatingdomains of GAL4 or the herpes virus simplex protein VP16.

[0124] The two fusion proteins are contained in a cell which alsocomprises a reporter gene. The reporter gene is sensitive to theactivation of the reconstituted sequence-specific transcriptionalactivating factor Suitable reporter genes whose expression can beconveniently detected include the E. coli lacZ gene, whose expressionmay be measured calorimetrically, and yeast selectable genes such asHIS3 or URA3

[0125] In the absence of the test compound, the cell expresses thereporter gene. A test compound is added to the cell, and the effect onexpression of the reporter gene is measured. A test compound whichdisrupts the binding of the respective binding domains of CIF130 andCIF150/hTAF_(II)150 will have a negative effect on the transcriptionalactivation ability of the reconstituted sequence-specifictranscriptional activating factor Thus, expression of the reporter genewill be decreased Compounds which decrease expression of the reportergene are potential inducers of mitosis or cell cycle progressionCompounds which increase expression of the reporter gene are potentialinhibitors of mitosis or cell cycle progression.

[0126] The CIF150/hTAF_(II)150-binding domain of CIF130 and theCIF130-binding domain of CIF150/hTAF_(II)150 can be readily determined,for example, by testing various portions of each protein for the abilityto bind to its partner A variety of techniques can be used for thispurpose, including but not limited to the yeast two-hybrid assay,affinity column chromatography, and polyacrylamide gel electrophoresisunder non-reducing conditions.

[0127] CIF130 subgenomic polynucleotides can also be delivered tosubjects for the purpose of screening for test compounds agents whichare useful for enhancing transfer of CIF130 subgenomic polynucleotidesto a cell, for example, by enhancing transfer of CIF130 subgenomicpolynucleotides to the cell or for enhancing subsequent biologicaleffects of the CIF130 subgenomic polynucleotides within the cell. Suchbiological effects include hybridization to complementary CIF130 mRNAand inhibition of its translation, expression of the CIF130 subgenomicpolynucleotide to form CIF130 mRNA and/or CIF130 protein, andreplication and integration of the CIF130 subgenomic polynucleotide Testcompounds which can be screened include any substances, whether naturalproducts or synthetic, which can be administered to the subject.Libraries or mixtures of compounds can be tested The compounds orsubstances can be those for which a pharmaceutical effect is previouslyknown or unknown. The compounds or substances can be delivered before,after, or concomitantly with the CIF130 subgenomic polynucleotides. Theycan be administered separately or in admixture with the CIF130subgenomic polynucleotides.

[0128] Integration of delivered CIF130 subgenomic polynucleotides can bemonitored by any means known in the art. For example, Southern blottingof the delivered CIF130 subgenomic polynucleotides can be performed. Achange in the size of the fragments of the delivered polynucleotidesindicates integration. Replication of the delivered polynucleotides canbe monitored infer alia by detecting incorporation of labelednucleotides combined with hybridization to a CIF130 probe. Expression ofa CIF130 subgenomic polynucleotide can be monitored by detectingproduction of CIF130 mRNA which hybridizes to the deliveredpolynucleotide or by detecting CIF130 protein. CIF130 protein can bedetected immunologically or by activity, for example by detectingbinding to CIF150/hTAF_(II)150. Thus, the delivery of CIF130 subgenomicpolynucleotides according to the present invention provides an excellentsystem for screening test compounds for their ability to enhancetransfer of CIF130 subgenomic polynucleotides to a cell, by enhancingdelivery, integration, hybridization, expression, replication orintegration in an animal, preferably a mammal, more preferably a human.

[0129] The complete contents of all references cited in this disclosureare incorporated herein by reference. The following is provided forexemplification purposes only and is not intended to limit the scope ofthe invention which has been described in broad terms above.

EXAMPLE 1

[0130] This example demonstrates purification of CIF130

[0131] Extracts of HeLa cells were prepared as described in Dignam etal., 1983, Nucl. Acids Res. 11, 1495-89 The 0.1 M KCl flowthroughfraction of a DEAE-Sephacel column was then applied to a Mono Q columnand eluted with a linear KCl gradient (40 ml; 0.1 to 1 M). TheCIF150/hTAF_(II)150/CIF130-containing fractions were pooled and dialyzedagainst buffer A (20 mM HEPES, pH 7.9, 1 mM EDTA, 3 mM dithiothreitol, 1mM phenylmethylsulfonyl fluoride, 20% glycerol) containing 0 1 M KCl.These fractions were supplemented with imidazole (final concentration,20 mM in buffer A) and applied to a Ni-nitrilotriacetic acid(NTA)-agarose column (Qiagen). After being washed with 10 column volumeseach of 20 mM imidazole and 35 mM imidazole, bound proteins were elutedwith 100 mM imidazole.

[0132] CIF130 copurifies with CIF150/hTAF_(II)150. PurifiedCIF150/hTAF_(II)150 and CIF 130 proteins were visualized by sodiumdodecyl sulfate-6% polyacrylamide gel electrophoresis, followed bysilver staining. CIF133was separated from CIF150/hTAF_(II)150 byexcising the CIF 130-containing band from the SDS gel and eluting theCIF130 protein.

EXAMPLE 2

[0133] This example demonstrates upregulation of CIF130 mRNA in humanbrain tumors.

[0134] Total RNA was isolated from human tumor and normal tissues fromfour different donors. The tissues included normal and tumor samples ofbreast, uterus, fallopian tube, and ovary, in addition to brain. Twentymicrograms of total RNA was loaded per lane and run on a 1% denaturingformaldehyde gel. Visual inspection of the gel after ethidium bromidestaining confirmed that there was less than 10% variation between thelanes. The gel was vacuum-blotted to a positively charged nylonmembrane, and the blot was fixed by UV irradiation and baking.

[0135] The nylon membrane was incubated with a radiolabeled CIF130probe. The results showed that CIF130 mRNA is upregulated in brain tumortissue compared with normal tissues from four different patients.

[0136] Those skilled in the art will recognize, or be able to ascertain,using not more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such specificembodiments and equivalents are intended to be encompassed by thefollowing claims.

[0137] All patents, published patent applications and publications citedherein are incorporated by reference as if set forth fully herein.

1 4 1 3825 DNA Homo sapien 1 gcttcccccg cctctccttc ctcgcgccgc cctggcccggccctgtcgcg catgaaaccc 60 gaggccctaa gcgaggcgcg ggcgccaaca tcgtcgacggcgacgtcggt atcgtcgtcc 120 agtcagtaac cgtggtactt gaccttattt ccaccaggaccgtgattcgc tcctaaaccg 180 aaacctccaa aacggtagtc acgacccttt ttcctccttgggtttgaggg tgtcgtcagg 240 gtgtcacgga aaccccgttg gtcgagaaga agacctaaacctttcagtcg aggcggtgtc 300 gaaggaagaa agatgtttta acctagattc gcccggttgaaactacttct tttacggata 360 aaacttctac tccttcttct aagatcgttg caactaaatggaatgtaagg acgacttttg 420 agtggttgag cggttgttaa ggtaaggttc ggtcatctaagactgtcgct actactaggg 480 aacctccgta agtaccgact ccacctccta gtccgccgatctctgtactt ctccgaactt 540 cttttcctgt tcctttcttt tttgcatttc ccataagctctactgtaact tctccttcta 600 ctggttcttc gaataaaagc tatgtaccgt cttttgggttgacgaccaca ccaagtcctc 660 cttctccttc tgttagatct tatactatca ctgcctttaggttaacgtgg atggtttttt 720 tagtaactag gagaaggggg gtaactagta agtctctaactgataggtgg taaacttttt 780 ttgaaaatgt tactcgtact tctctattgg ttggagtgaggtgtcgtcaa ttatctagag 840 gccgtattcg agttagaagc ccagagacca cgacgtggaggatctggtcc ttcatcgaaa 900 cgagtaaaac ccaaactgct tgttgaatac gtggtctaagcctttagact tatgtgtgtc 960 gggtgaggtt atgtcacggt cccacacgga caccgtaattcaccatctct gtactaacca 1020 taacggtttt gtccatcacc cttttgacgt cggaagtaaaccgggtacaa ctaagtatat 1080 tacctggtct tcctcaacct tggtccacta cctggttaacgtcactaaca cacaggatgg 1140 tccctcgaaa cggtcgtcta ggtaggtctt acattcgccaaaccttttcg tatattagaa 1200 gctagtcacc ggcatatacc tcctccctca tacaccctcgtccggttccg ggaagtcctc 1260 ccccgtctct aacaacacac atggggtcca gctgactatctagtacactt tttctttcga 1320 tggttagaag tttctcagag aatggaacac aaactacttcgtctagctta caaactgtac 1380 cctaaactca tggttcaagc taggtatcgt tcagtacaagcaggactgtc cgtctgagag 1440 aataaatcac gttgaaaagc cttcttctaa cttttcaaccggtctctgta ggactagctg 1500 ggataagctc accacgtccc tctataacct ctccgtttacttctacactg tgtctaacac 1560 ctctaagagg taagacctgg atcatttacc ttgaccgaatgggccgcaga ccatcttaaa 1620 tggagaagtc cctcacagga ggagaaacaa tgattttttcggttacgact tctcgatcgc 1680 ttattggaat ttgtcctccc agtattagaa cccgacgaggtacccctata cctagtctca 1740 ctctctttgt tccagtaaag tctgaaattc tttttcctgtagggtcagga ccaccggtgt 1800 ctacaacgtc gggcaccaga cctgtaagga agttaattctgacagtaatt gatactacac 1860 cgtgccctgt aactatgcgt gtgagtgtcc taaccggcgtgtccttctcg cccactcttt 1920 ccacaccgga tatgggatga gtgagggttc ctgtcgttaaaacgaccact ggaccaggcc 1980 ttgaaccttc ctcggttagt tgtgcaaaga ttccttgaggatctagaccg ttacgtctta 2040 cggaccaaag cctttagagc taagtttcct ccctttccttttttcgactt gtaaccacct 2100 cctccggatc cgatgtccct cgccggaccg gacccgagactcttgtacct agctccttta 2160 ttgttacatt actcgttaat actccggatg ttcggaaggtgtcctcgata ccctctagct 2220 gattgccgtt actttcgtcg aaaggtcagt gtcatgttctcagtgaaaca acgtcggtca 2280 aattcattag tcttccgacc ttcaagacga cggccccgttcacccacctg atcacgtccc 2340 tcgaacttaa gacaaggttg attgagtcgt gttgtcccggtattgtcagg actgtcgggg 2400 cagtggtcac ggcggttccc gtagggtccg aaaccgttatgaccgttgta gtcaccacgg 2460 ggacactgga tgggcagacg gcctcgggtt cctcagttgttgtgtcgaag tcccttattg 2520 tcggctcttc cctgaccccc gtcgttgccc ttttccctctctatatgact cttggccccg 2580 tcgtcggcag tgtcagtgcc tctctgaccg ttagccgtatcgctatcagg tgcagtgcct 2640 ctaccaccag cggtacctct acctatggcg gtaggtctttcgtcgtcggc agtatgacta 2700 ccggtagccg tgcccctctt gtctgtacct ccttcgcgtccggccgtacc cctcttggcc 2760 ccacgtttac taccagcctt acccctttcg tcctttcttcgaaaattagc actcccgttc 2820 tacctcgggt tctaccttgg gtttcgcctg tcgtcgttctacctgttcca cctgtcgttc 2880 tgtctattct gtcgactgcc aaaacgacag ggtctcggcggatttgcgtt ctttccagct 2940 accctgtcaa tctcccctac acgatttcgc actttagtcaacaggaatta aaaatctttc 3000 taaaaccatt gatccacaga gtcccgaccc aaccccaggtttcacattcc tgggggacgg 3060 gaatcacctc tcgacctcga acctctgtaa tggggaagtagtcttcctta aaagcctaca 3120 aaagaaccct tcgacaaaac caggaacctt cgtcactctcgacccttcga agaaaaccga 3180 gatccactca acagtacgcc cattcaactc caatagaaccctatttccca gaagatcccg 3240 tgttttgagt gagatccaaa tataatatac atcgaatataaaaaatgatt ccacagtgga 3300 atattcgtag atatttaact caagaaaaag aatcaacataccggtccgtc aggggtaaaa 3360 tcctcaaccg aagacgttta agttaggtaa ctcgattgacaacccctcgt taaaccatca 3420 acatctgtaa acgtcccttc cctctacaga ctaagatttaccctcaacta cgagtccagg 3480 ggtcggtcca aacgtaggtc gggactctgt acatcctttgtggaaagtct gggtccgaga 3540 cttctaaggg tcttcggtgt tcctaacttc ccttttccactaggaccatt gacaaggtcc 3600 taacgaggtc caaactctac cataacgatt taaattttaatttgttctct gggttgttgt 3660 cgaaaatttc acagaagata aagtaacata aaaaaaattgaacggggtta ctatcttttc 3720 agaaaacgac tttactaaaa ctactaaaaa caaatagcaaatatttttcc ttttctttat 3780 attttttttt tttttttttt tttttttttt cgccggcgacttaag 3825 2 1261 PRT Homo sapien 2 Arg Arg Gly Arg Arg Gly Arg Ser AlaAla Gly Pro Gly Arg Asp Ser 1 5 10 15 Ala Tyr Phe Gly Leu Arg Asp SerLeu Arg Ala Arg Gly Cys Ser Ser 20 25 30 Cys Arg Cys Ser His Ser Ser ArgSer Val Ile Gly Thr Met Asn Trp 35 40 45 Asn Lys Gly Gly Pro Gly Thr LysArg Gly Phe Gly Phe Gly Gly Phe 50 55 60 Ala Ile Ser Ala Gly Lys Lys GluGlu Pro Lys Leu Pro Gln Gln Ser 65 70 75 80 His Ser Ala Phe Gly Ala ThrSer Ser Ser Ser Gly Phe Gly Lys Ser 85 90 95 Ala Pro Pro Gln Leu Pro SerPhe Tyr Lys Ile Gly Ser Lys Arg Ala 100 105 110 Asn Phe Asp Glu Glu AsnAla Tyr Phe Glu Asp Glu Glu Glu Asp Ser 115 120 125 Ser Asn Val Asp LeuPro Tyr Ile Pro Ala Glu Asn Ser Pro Thr Arg 130 135 140 Gln Gln Phe HisSer Lys Pro Val Asp Ser Asp Ser Asp Asp Asp Pro 145 150 155 160 Leu GluAla Phe Met Ala Glu Val Glu Asp Gln Ala Ala Arg Asp Met 165 170 175 LysArg Leu Glu Glu Lys Asp Lys Glu Arg Lys Asn Val Lys Gly Ile 180 185 190Arg Asp Asp Ile Glu Glu Glu Asp Asp Gln Glu Ala Tyr Phe Arg Tyr 195 200205 Met Ala Glu Asn Pro Thr Ala Gly Val Val Gln Glu Glu Glu Glu Asp 210215 220 Asn Leu Glu Tyr Asp Ser Asp Gly Asn Pro Ile Ala Pro Thr Lys Lys225 230 235 240 Ile Ile Asp Pro Leu Pro Pro Ile Asp His Ser Glu Ile AspTyr Pro 245 250 255 Pro Phe Glu Lys Asn Phe Tyr Asn Glu His Glu Glu IleThr Asn Leu 260 265 270 Thr Pro Gln Gln Leu Ile Asp Leu Arg His Lys LeuAsn Leu Arg Val 275 280 285 Ser Gly Ala Ala Pro Pro Arg Pro Gly Ser SerPhe Ala His Phe Gly 290 295 300 Phe Asp Glu Gln Leu Met His Gln Ile ArgLys Ser Glu Tyr Thr Gln 305 310 315 320 Pro Thr Pro Ile Gln Cys Gln GlyVal Pro Val Ala Leu Ser Gly Arg 325 330 335 Asp Met Ile Gly Ile Ala LysThr Gly Ser Gly Lys Thr Ala Ala Phe 340 345 350 Ile Trp Pro Met Leu IleHis Ile Met Asp Gln Lys Glu Leu Glu Pro 355 360 365 Gly Asp Gly Pro IleAla Val Ile Val Cys Pro Thr Arg Glu Leu Cys 370 375 380 Gln Gln Ile HisPro Glu Cys Lys Arg Phe Gly Lys Ala Tyr Asn Leu 385 390 395 400 Arg SerVal Ala Val Tyr Gly Gly Gly Ser Met Trp Glu Gln Ala Lys 405 410 415 AlaLeu Gln Glu Gly Ala Glu Ile Val Val Cys Thr Pro Gly Arg Leu 420 425 430Ile Asp His Val Lys Lys Lys Ala Thr Asn Leu Gln Arg Val Ser Tyr 435 440445 Leu Val Phe Asp Glu Ala Asp Arg Met Phe Asp Met Gly Phe Glu Tyr 450455 460 Gln Val Arg Ser Ile Ala Ser His Val Arg Pro Asp Arg Gln Thr Leu465 470 475 480 Leu Phe Ser Ala Thr Phe Arg Lys Lys Ile Glu Lys Leu AlaArg Asp 485 490 495 Ile Leu Ile Asp Pro Ile Arg Val Val Gln Gly Asp IleGly Glu Ala 500 505 510 Asn Glu Asp Val Thr Gln Ile Val Glu Ile Leu HisSer Gly Pro Ser 515 520 525 Lys Trp Asn Trp Leu Thr Arg Arg Leu Val GluPhe Thr Ser Ser Gly 530 535 540 Ser Val Leu Leu Phe Val Thr Lys Lys AlaAsn Ala Glu Glu Leu Ala 545 550 555 560 Asn Asn Leu Lys Gln Glu Gly HisAsn Leu Gly Leu Leu His Gly Asp 565 570 575 Met Asp Gln Ser Glu Arg AsnLys Val Ile Ser Asp Phe Lys Lys Lys 580 585 590 Asp Ile Pro Val Leu ValAla Thr Asp Val Ala Ala Arg Gly Leu Asp 595 600 605 Ile Pro Ser Ile LysThr Val Ile Asn Tyr Asp Val Ala Arg Asp Ile 610 615 620 Asp Thr His ThrHis Arg Ile Gly Arg Thr Gly Arg Ala Gly Glu Lys 625 630 635 640 Gly ValAla Tyr Thr Leu Leu Thr Pro Lys Asp Ser Asn Phe Ala Gly 645 650 655 AspLeu Val Arg Asn Leu Glu Gly Ala Asn Gln His Val Ser Lys Glu 660 665 670Leu Leu Asp Leu Ala Met Gln Asn Ala Trp Phe Arg Lys Ser Arg Phe 675 680685 Lys Gly Gly Lys Gly Lys Lys Leu Asn Ile Gly Gly Gly Gly Leu Gly 690695 700 Tyr Arg Glu Arg Pro Gly Leu Gly Ser Glu Asn Met Asp Arg Gly Asn705 710 715 720 Asn Asn Val Met Ser Asn Tyr Glu Ala Tyr Lys Pro Ser ThrGly Ala 725 730 735 Met Gly Asp Arg Leu Thr Ala Met Lys Ala Ala Phe GlnSer Gln Tyr 740 745 750 Lys Ser His Phe Val Ala Ala Ser Leu Ser Asn GlnLys Ala Gly Ser 755 760 765 Ser Ala Ala Gly Ala Ser Gly Trp Thr Ser AlaGly Ser Leu Asn Ser 770 775 780 Val Pro Thr Asn Ser Ala Gln Gln Gly HisAsn Ser Pro Asp Ser Pro 785 790 795 800 Val Thr Ser Ala Ala Lys Gly IlePro Gly Phe Gly Asn Thr Gly Asn 805 810 815 Ile Ser Gly Ala Pro Val ThrTyr Pro Ser Ala Gly Ala Gln Gly Val 820 825 830 Asn Asn Thr Ala Ser GlyAsn Asn Ser Arg Glu Gly Thr Gly Gly Ser 835 840 845 Asn Gly Lys Arg GluArg Tyr Thr Glu Asn Arg Gly Ser Ser Arg His 850 855 860 Ser His Gly GluThr Gly Asn Arg His Ser Asp Ser Pro Arg His Gly 865 870 875 880 Asp GlyGly Arg His Gly Asp Gly Tyr Arg His Pro Glu Ser Ser Ser 885 890 895 ArgHis Thr Asp Gly His Arg His Gly Glu Asn Arg His Gly Gly Ser 900 905 910Ala Gly Arg His Gly Glu Asn Arg Gly Ala Asn Asp Gly Arg Asn Gly 915 920925 Glu Ser Arg Lys Glu Ala Phe Asn Arg Glu Gly Lys Met Glu Pro Lys 930935 940 Met Glu Pro Lys Ala Asp Ser Ser Lys Met Asp Lys Val Asp Ser Lys945 950 955 960 Thr Asp Lys Thr Ala Asp Gly Phe Ala Val Pro Glu Pro ProLys Arg 965 970 975 Lys Lys Gly Arg Trp Asp Ser Arg Gly Cys Ala Lys AlaAsn Gln Leu 980 985 990 Ser Leu Ile Phe Arg Lys Ile Leu Val Thr Arg CysLeu Arg Ala Gly 995 1000 1005 Leu Gly Ser Lys Val Gly Pro Pro Ala LeuSer Gly Glu Leu Glu Leu 1010 1015 1020 Gly Asp Ile Thr Pro Ser Ser GluGly Ile Phe Gly Cys Phe Leu Gly 1025 1030 1035 1040 Lys Leu Phe Trp SerLeu Glu Ala Val Arg Ala Gly Lys Leu Leu Leu 1045 1050 1055 Ala Leu GlyGlu Leu Ser Cys Gly Val Glu Val Ile Leu Gly Arg Val 1060 1065 1070 PheGly Thr Lys Leu Thr Leu Gly Leu Tyr Tyr Met Leu Ile Phe Phe 1075 10801085 Thr Lys Val Ser Pro Tyr Lys His Leu Ile Glu Phe Phe Phe Leu Val1090 1095 1100 Val Trp Pro Gly Ser Pro His Phe Arg Ser Trp Leu Leu GlnIle Gln 1105 1110 1115 1120 Ser Ile Glu Leu Thr Val Gly Glu Gln Phe GlySer Cys Arg His Leu 1125 1130 1135 Gln Gly Arg Glu Met Ser Asp Ser LysTrp Glu Leu Met Leu Arg Ser 1140 1145 1150 Pro Ala Arg Phe Ala Ser SerPro Glu Thr Cys Arg Lys His Leu Ser 1155 1160 1165 Asp Pro Gly Ser GluAsp Ser Gln Lys Pro Gln Gly Leu Lys Gly Lys 1170 1175 1180 Gly Asp ProGly Asn Cys Ser Arg Ile Ala Pro Gly Leu Arg Trp Tyr 1185 1190 1195 1200Cys Ile Asn Thr Arg Asp Pro Thr Thr Ala Phe Lys Val Ser Ser Ile 12051210 1215 Ser Leu Tyr Phe Phe Leu Ala Pro Met Ile Glu Lys Ser Phe AlaGlu 1220 1225 1230 Met Ile Leu Met Ile Phe Val Tyr Arg Leu Lys Gly LysGlu Ile Lys 1235 1240 1245 Lys Lys Lys Lys Lys Lys Lys Lys Ala Ala AlaGlu Phe 1250 1255 1260 3 3996 DNA Homo sapien 3 caagatgtcg gcggatggtagcttcgagcc cttgcggaga ggagcatctc tgtgacagaa 60 gcttgtcgac ggcggcttctaggagctagt cgaaggagcg aggttgaggc gggcagcgac 120 ccgtcaggtc gctcacctgggcaccggcca gctgcgagac gtgacttggg gaccgcaggg 180 gagtggagag tgtgaggtgccaaagactag taatgccccg tatcccccta ggaagccggg 240 aagccaagct ccgcgggaccgcttcatgcc gctgactggt gtagagcccg ccagaatgaa 300 caggaagaaa ggagacaagggctttgaaag cccaaggcca tataaattaa cccatcaggt 360 cgtctgcatc aacaacataaatttccacag aaaatctgtt gtgggatttg tggaactgac 420 tatattcccc acagttgcaaacttgaatag aatcaagttg aacagcaaac agtgtagaat 480 ataccgagta aggatcaatgatttagaggc tgcttttatt tataatgacc caaccttgga 540 agtttgtcac agtgaatcaaaacagagaaa cctcaattat ttttccaatg cttatgcagc 600 tgcagttagt gctgtggaccctgatgcagg aaatggagaa ctttgcatta aggttccatc 660 agagctatgg aaacacgttgatgagttaaa ggtcctgaag atacacatca atttttcttt 720 ggatcagccc aaaggaggtcttcattttgt ggtacccagt gtagagggaa gtatggcaga 780 gagaggtgct catgttttctcttgtgggta tcaaaattct acaagatttt ggttcccttg 840 tgttgattca tactctgaattgtgtacatg gaaattagaa tttacagtag atgctgcaat 900 ggttgctgtt tctaatggcgatttggtgga gacagtgtat actcatgata tgaggaagaa 960 aactttccat tatatgcttaccattcctac agcagcgtca aatatctcct tggccattgg 1020 accatttgaa atactggtagatccatacat gcatgaggtt actcattttt gtttgcccca 1080 acttcttcca ttgctgaaacataccacatc ataccttcat gaagtctttg aattttatga 1140 agaaattctt acatgtcgttacccatactc ctgttttaag actgtcttca ttgatgaggc 1200 ttatgttgaa gtggctgcttatgcttccat gagcattttt agcacaaatc ttttacacag 1260 tgccatgatt atagatgagacacctttgac tagaaggtgt ttagcccaat ccttggccca 1320 gcagtttttt ggttgtttcatatctagaat gtcttggtct gatgaatggg tgctgaaggg 1380 aatttcaggc tatatctatggactttggat gaaaaaaact tttggtgtta atgagtaccg 1440 ccattggatt aaagaggagctagacaaaat agtggcatat gaactaaaaa ctggtggggt 1500 tttactacat cccatatttggtggaggaaa agagaaggat aatccggctt cccatctaca 1560 cttttcaata aagcatccacatacactgtc ctgggaatac tacactatgt ttcagtgtaa 1620 agcccacctt gtgatgagattgattgaaaa taggatcagt atggaattta tgctacaagt 1680 tttcaataaa ctgctaagtctggctagtac tgcttcatct cagaagttcc agtcacatat 1740 gtggagtcag atgttggtttccacatctgg gtttttgaaa tccatttcaa atgtctctgg 1800 caaagatatt cagccgttaataaagcagtg ggtagatcag agtggagtgg taaaatttta 1860 tggaagtttt gcatttaatagaaaacgaaa tgtcttggaa ctggaaataa aacaggacta 1920 tacatctcct ggaactcagaaatacgtggg accacttaaa gtgacagtgc aggagttaga 1980 tggatccttc aatcatacactgcaaattga agaaaacagc cttaaacatg atataccctg 2040 ccattccaaa agtagaaggaataaaaagaa aaaaatccca ctgatgaatg gagaagaagt 2100 tgatatggat ctttctgcaatggatgctga ttcccctttg ctgtggataa ggatagaccc 2160 agatatgtca gtattgaggaaggtagaatt tgagcaagct gattttatgt ggcagtatca 2220 gctccgctat gagagagatgttgttgcaca gcaggaatcc attttggctt tggaaaaatt 2280 ccctactcca gcatctcggcttgcactcac tgatatatta gaacaagagc agtgtttcta 2340 cagagtaaga atgtcagcttgtttctgtct tgcaaagatt gcaaattcaa tggtgagcac 2400 atggacagga ccaccagccatgaagtcact cttcactagg atgttttgtt gtaaaagttg 2460 tccaaacatt gtgaaaacaaacaactttat gagctttcaa agctattttc tacagaagac 2520 tatgccagtt gcaatggctttattaagaga tgttcataat ctttgtccta aagaagtctt 2580 aacatttatt ttagacttaatcaagtacaa tgacaacagg aaaaataagt tttcagataa 2640 ctattatcgt gcagaaatgattgatgccct ggccaactct gttacacctg cagtcagtgt 2700 gaataatgaa gttagaactttggataactt aaatcctgat gtgcgactca ttcttgaaga 2760 aatcaccaga tttttgaatatggaaaaact tcttccgagt tacaggcata ccatcactgt 2820 cagttgtttg agagccatacgggtacttca gaagaacgga catgtgccaa gtgatccagc 2880 tctttttaaa tcttatgctgaatatggcca ctttgtggac attaggatag cagctttgga 2940 agcagttgtt gattatactaaagtggacag aagttatgaa gaactgcaat ggctacttaa 3000 tatgattcag aatgaccctgtaccctatgt aaggcataag attctcaaca tgttgactaa 3060 gaacccccca tttactaagaacatggagtc tcccttatgc aatgaagccc tggtagatca 3120 actttggaaa cttatgaattctggtacttc acatgactgg aggttacggt gtggtgctgt 3180 ggacttgtac ttcacactttttggcctcag tagaccttcc tgtttaccct tgccagagct 3240 tgggttggtt cttaatctaaaggagaaaaa agctgtcttg aatcctacca taattccaga 3300 gtcagtagca ggcaaccaagaagctgcaaa taatccaagc agtcacccac agctagttgg 3360 atttcagaac cctttttccagttctcaaga tgaggaggag attgatatgg atactgttca 3420 tgatagccag gccttcatttcccatcattt aaacatgctt gaaaggccgt caactccagg 3480 gctctcgaaa tatcggccagctagctcccg atctgcttta ataccccagc actcagcagg 3540 ctgtgacagc acacccaccacaaaacccca gtggagtttg gaacttgcac ggaagggaac 3600 aggtaaagaa caagcacctttggagatgag tatgcatcca gcggcaagcg ctccactctc 3660 agtctttact aaggaatctacagcctccaa acacagtgac caccatcacc accatcacca 3720 tgagcacaag aaaaagaagaagaagcataa acataagcac aaacacaagc ataagcatga 3780 cagtaaagaa aaggacaaggagcctttcac tttctccagc cctgccagtg gcaggtctat 3840 tcgttctcct tccctttcagactgagaagg ggacaaaaag acctttcctt tcatgtccag 3900 aagaatgtat gtaactaaagctttgtcctc tgtgaagaat tataaatgga ggggggaaag 3960 gattcgcctc tcctacagaaattctgaatt cattta 3996 4 1199 PRT Homo sapien 4 Met Pro Leu Thr Gly ValGlu Pro Ala Arg Met Asn Arg Lys Lys Gly 1 5 10 15 Asp Lys Gly Phe GluSer Pro Arg Pro Tyr Lys Leu Thr His Gln Val 20 25 30 Val Cys Ile Asn AsnIle Asn Phe His Arg Lys Ser Val Val Gly Phe 35 40 45 Val Glu Leu Thr IlePhe Pro Thr Val Ala Asn Leu Asn Arg Ile Lys 50 55 60 Leu Asn Ser Lys GlnCys Arg Ile Tyr Arg Val Arg Ile Asn Asp Leu 65 70 75 80 Glu Ala Ala PheIle Tyr Asn Asp Pro Thr Leu Glu Val Cys His Ser 85 90 95 Glu Ser Lys GlnArg Asn Leu Asn Tyr Phe Ser Asn Ala Tyr Ala Ala 100 105 110 Ala Val SerAla Val Asp Pro Asp Ala Gly Asn Gly Glu Leu Cys Ile 115 120 125 Lys ValPro Ser Glu Leu Trp Lys His Val Asp Glu Leu Lys Val Leu 130 135 140 LysIle His Ile Asn Phe Ser Leu Asp Gln Pro Lys Gly Gly Leu His 145 150 155160 Phe Val Val Pro Ser Val Glu Gly Ser Met Ala Glu Arg Gly Ala His 165170 175 Val Phe Ser Cys Gly Tyr Gln Asn Ser Thr Arg Phe Trp Phe Pro Cys180 185 190 Val Asp Ser Tyr Ser Glu Leu Cys Thr Trp Lys Leu Glu Phe ThrVal 195 200 205 Asp Ala Ala Met Val Ala Val Ser Asn Gly Asp Leu Val GluThr Val 210 215 220 Tyr Thr His Asp Met Arg Lys Lys Thr Phe His Tyr MetLeu Thr Ile 225 230 235 240 Pro Thr Ala Ala Ser Asn Ile Ser Leu Ala IleGly Pro Phe Glu Ile 245 250 255 Leu Val Asp Pro Tyr Met His Glu Val ThrHis Phe Cys Leu Pro Gln 260 265 270 Leu Leu Pro Leu Leu Lys His Thr ThrSer Tyr Leu His Glu Val Phe 275 280 285 Glu Phe Tyr Glu Glu Ile Leu ThrCys Arg Tyr Pro Tyr Ser Cys Phe 290 295 300 Lys Thr Val Phe Ile Asp GluAla Tyr Val Glu Val Ala Ala Tyr Ala 305 310 315 320 Ser Met Ser Ile PheSer Thr Asn Leu Leu His Ser Ala Met Ile Ile 325 330 335 Asp Glu Thr ProLeu Thr Arg Arg Cys Leu Ala Gln Ser Leu Ala Gln 340 345 350 Gln Phe PheGly Cys Phe Ile Ser Arg Met Ser Trp Ser Asp Glu Trp 355 360 365 Val LeuLys Gly Ile Ser Gly Tyr Ile Tyr Gly Leu Trp Met Lys Lys 370 375 380 ThrPhe Gly Val Asn Glu Tyr Arg His Trp Ile Lys Glu Glu Leu Asp 385 390 395400 Lys Ile Val Ala Tyr Glu Leu Lys Thr Gly Gly Val Leu Leu His Pro 405410 415 Ile Phe Gly Gly Gly Lys Glu Lys Asp Asn Pro Ala Ser His Leu His420 425 430 Phe Ser Ile Lys His Pro His Thr Leu Ser Trp Glu Tyr Tyr ThrMet 435 440 445 Phe Gln Cys Lys Ala His Leu Val Met Arg Leu Ile Glu AsnArg Ile 450 455 460 Ser Met Glu Phe Met Leu Gln Val Phe Asn Lys Leu LeuSer Leu Ala 465 470 475 480 Ser Thr Ala Ser Ser Gln Lys Phe Gln Ser HisMet Trp Ser Gln Met 485 490 495 Leu Val Ser Thr Ser Gly Phe Leu Lys SerIle Ser Asn Val Ser Gly 500 505 510 Lys Asp Ile Gln Pro Leu Ile Lys GlnTrp Val Asp Gln Ser Gly Val 515 520 525 Val Lys Phe Tyr Gly Ser Phe AlaPhe Asn Arg Lys Arg Asn Val Leu 530 535 540 Glu Leu Glu Ile Lys Gln AspTyr Thr Ser Pro Gly Thr Gln Lys Tyr 545 550 555 560 Val Gly Pro Leu LysVal Thr Val Gln Glu Leu Asp Gly Ser Phe Asn 565 570 575 His Thr Leu GlnIle Glu Glu Asn Ser Leu Lys His Asp Ile Pro Cys 580 585 590 His Ser LysSer Arg Arg Asn Lys Lys Lys Lys Ile Pro Leu Met Asn 595 600 605 Gly GluGlu Val Asp Met Asp Leu Ser Ala Met Asp Ala Asp Ser Pro 610 615 620 LeuLeu Trp Ile Arg Ile Asp Pro Asp Met Ser Val Leu Arg Lys Val 625 630 635640 Glu Phe Glu Gln Ala Asp Phe Met Trp Gln Tyr Gln Leu Arg Tyr Glu 645650 655 Arg Asp Val Val Ala Gln Gln Glu Ser Ile Leu Ala Leu Glu Lys Phe660 665 670 Pro Thr Pro Ala Ser Arg Leu Ala Leu Thr Asp Ile Leu Glu GlnGlu 675 680 685 Gln Cys Phe Tyr Arg Val Arg Met Ser Ala Cys Phe Cys LeuAla Lys 690 695 700 Ile Ala Asn Ser Met Val Ser Thr Trp Thr Gly Pro ProAla Met Lys 705 710 715 720 Ser Leu Phe Thr Arg Met Phe Cys Cys Lys SerCys Pro Asn Ile Val 725 730 735 Lys Thr Asn Asn Phe Met Ser Phe Gln SerTyr Phe Leu Gln Lys Thr 740 745 750 Met Pro Val Ala Met Ala Leu Leu ArgAsp Val His Asn Leu Cys Pro 755 760 765 Lys Glu Val Leu Thr Phe Ile LeuAsp Leu Ile Lys Tyr Asn Asp Asn 770 775 780 Arg Lys Asn Lys Phe Ser AspAsn Tyr Tyr Arg Ala Glu Met Ile Asp 785 790 795 800 Ala Leu Ala Asn SerVal Thr Pro Ala Val Ser Val Asn Asn Glu Val 805 810 815 Arg Thr Leu AspAsn Leu Asn Pro Asp Val Arg Leu Ile Leu Glu Glu 820 825 830 Ile Thr ArgPhe Leu Asn Met Glu Lys Leu Leu Pro Ser Tyr Arg His 835 840 845 Thr IleThr Val Ser Cys Leu Arg Ala Ile Arg Val Leu Gln Lys Asn 850 855 860 GlyHis Val Pro Ser Asp Pro Ala Leu Phe Lys Ser Tyr Ala Glu Tyr 865 870 875880 Gly His Phe Val Asp Ile Arg Ile Ala Ala Leu Glu Ala Val Val Asp 885890 895 Tyr Thr Lys Val Asp Arg Ser Tyr Glu Glu Leu Gln Trp Leu Leu Asn900 905 910 Met Ile Gln Asn Asp Pro Val Pro Tyr Val Arg His Lys Ile LeuAsn 915 920 925 Met Leu Thr Lys Asn Pro Pro Phe Thr Lys Asn Met Glu SerPro Leu 930 935 940 Cys Asn Glu Ala Leu Val Asp Gln Leu Trp Lys Leu MetAsn Ser Gly 945 950 955 960 Thr Ser His Asp Trp Arg Leu Arg Cys Gly AlaVal Asp Leu Tyr Phe 965 970 975 Thr Leu Phe Gly Leu Ser Arg Pro Ser CysLeu Pro Leu Pro Glu Leu 980 985 990 Gly Leu Val Leu Asn Leu Lys Glu LysLys Ala Val Leu Asn Pro Thr 995 1000 1005 Ile Ile Pro Glu Ser Val AlaGly Asn Gln Glu Ala Ala Asn Asn Pro 1010 1015 1020 Ser Ser His Pro GlnLeu Val Gly Phe Gln Asn Pro Phe Ser Ser Ser 1025 1030 1035 1040 Gln AspGlu Glu Glu Ile Asp Met Asp Thr Val His Asp Ser Gln Ala 1045 1050 1055Phe Ile Ser His His Leu Asn Met Leu Glu Arg Pro Ser Thr Pro Gly 10601065 1070 Leu Ser Lys Tyr Arg Pro Ala Ser Ser Arg Ser Ala Leu Ile ProGln 1075 1080 1085 His Ser Ala Gly Cys Asp Ser Thr Pro Thr Thr Lys ProGln Trp Ser 1090 1095 1100 Leu Glu Leu Ala Arg Lys Gly Thr Gly Lys GluGln Ala Pro Leu Glu 1105 1110 1115 1120 Met Ser Met His Pro Ala Ala SerAla Pro Leu Ser Val Phe Thr Lys 1125 1130 1135 Glu Ser Thr Ala Ser LysHis Ser Asp His His His His His His His 1140 1145 1150 Glu His Lys LysLys Lys Lys Lys His Lys His Lys His Lys His Lys 1155 1160 1165 His LysHis Asp Ser Lys Glu Lys Asp Lys Glu Pro Phe Thr Phe Ser 1170 1175 1180Ser Pro Ala Ser Gly Arg Ser Ile Arg Ser Pro Ser Leu Ser Asp 1185 11901195

1. An isolated and purified human CIF130 protein comprising an aminoacid sequence which is at least 85% identical to the amino acid sequenceshown in SEQ ID NO: 2, wherein percent identity is determined using aSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of
 1. 2. Theisolated human CIF130 protein of claim 1 which has the amino acidsequence shown in SEQ ID NO:
 2. 3. The protein of claim 1 comprising atleast 17 contiguous amino acids as shown in SEQ ID NO:
 2. 4. A CIF130fusion protein comprising a first protein segment and a second proteinsegment fused together by means of a peptide bond, wherein the firstprotein segment consists of the polypeptide of claim
 3. 5. A preparationof antibodies which specifically binds to the protein of claim
 2. 6. AcDNA molecule which enclodes the protein of claim
 1. 7. The cDNAmolecule of claim 6 which encodes at least 17 contiguous amino acids ofSEQ ID NO:
 2. 8. The cDNA molecule of claim 7 which encodes SEQ ID NO:2.
 9. The cDNA molecule of claim 8 which comprises SEQ ED NO:
 1. 10. ThecDNA molecule of claim 6 comprising at least 12 contiguous nucleotidesof SEQ ID NO:
 1. 11. A cDNA molecule which is at least 85% identical tothe nucleotide sequence shown in SEQ ID NO: 1, wherein percent identityis determined using a Smith-Waterman homology search algorithm asimplemented in a MPSRCH program using an affine gap search with a gapopen penalty of 12 and a gap extension penalty of
 1. 12. An isolated andpurified subgenomic polynucleotide comprising a nucleotide sequencewhich hybridizes to SEQ ID NO: 1 after washing with 0.2×SSC at 65° C.,wherein the nucleotide sequence encodes a CIF130 protein having theamino acid sequence of SEQ ID NO:
 2. 13. A construct comprising: apromoter; and a polynucleotide segment encoding at least 17 contiguousamino acids of a human CIF130 protein as shown in SEQ ID NO: 2, whereinthe polynucleotide segment is located downstream from the promoter,wherein transcription of the polynucleotide segment initiates at thepromoter.
 14. A host cell comprising the construct of claim 13
 15. Ahomologously recombinant cell having incorporated therein a newtranscription initiation unit, wherein the new transcription initiationunit comprises: (a) an exogenous regulatory sequence; (b) an exogenousexon; and (c) a splice donor site, wherein the transcription initiationunit is located upstream of a coding sequence of a CIF130 gene as shownin SEQ ID NO: 1, wherein the exogenous regulatory sequence directstranscription of the coding sequence of the CIF130 gene.
 16. Thehomologously recombinant cell of claim 15 wherein the exogenousregulatory sequence directs enhanced transcription of the codingsequence of the CIF130 gene.
 17. A method to aid in the diagnosis orprognosis of neoplasia in a human, comprising the step of: comparingexpression of a first CIF130 gene in a first tissue of a human suspectedof being neoplastic with expression of a second CIF130 gene in a secondtissue of a human which is normal, wherein increased expression of thefirst CIF130 gene relative to expression of the second CIF130 geneindicates neoplasia in the first tissue.
 18. A method to aid in thediagnosis or prognosis of neoplasia in a human, comprising the step of:comparing a first human CIF130 gene, mRNA, or protein in a first tissuesuspected of being neoplastic with a second human CIF130 gene, mRNA, orprotein in a second tissue which is normal, wherein the second CIF130gene has a coding sequence as shown in SEQ ID NO: 1, wherein adifference between the first and second CIF130 genes, mRNAs, or proteinsindicates neoplasia in the first tissue.
 19. A method to aid indetecting a genetic predisposition to neoplasia in a human, comprising:comparing a CIF130 gene, mRNA, or protein in a fetal tissue of a humanwith a wild-type human CIF130 gene, mRNA, or protein, wherein thewild-type human CIF130 gene has a coding sequence as shown in SEQ ID NO:1, wherein a difference between the CIF130 gene, mRNA, or protein in thefetal tissue of the human and the wild-type human CIF130 gene, mRNA, orprotein indicates a genetic predisposition to neoplasia in the human.20. A method of screening test compounds for the ability to interferewith the binding of a CIF130 protein to a CIF150/hTAF_(II)150 protein,comprising the steps of: (a) contacting a test compound with at least aCIF150/hTAF_(II)150-binding domain of a CIF130 protein as shown in SEQID NO: 2 and at least a CIF130-binding domain of a CIF150/hTAF_(II)150protein as shown in SEQ ID NO: 4, wherein theCIF150/hTAF_(II)150-binding domain binds to the CIF130-binding domain inthe absence of the test compound, and (b) determining the amount of theCIF150/hTAF_(II)150-binding domain which is bound or unbound to theCIF130-binding domain or determining the amount of the CIF130-bindingdomain which is bound or unbound to the CIF150/hTAF_(II)150-bindingdomain in the presence of the test compound, wherein a test compoundwhich decreases the amount of bound CIF150/hTAF_(II)150- orCIF130-binding domains or which increases the amount of unboundCIF150/hTAF_(II)150- and CIF130-binding domains is a potential inducerof mitosis or cell cycle progression.
 21. A method of screening testcompounds for the ability to interfere with the binding of a CIF130protein to a CIF150/hTAF_(II)150 protein, comprising the steps of: (a)contacting a cell with a test compound, wherein the cell comprises: i) afirst fusion protein comprising (1) a CIF150/hTAF_(II)150-binding domainof a CIF130 protein as shown in SEQ ID NO: 2 and (2) either a DNAbinding domain or a transcriptional activating domain; ii) a secondfusion protein comprising a CIF130-binding domain of aCIF150/hTAF_(II)150 protein as shown in SEQ ID NO: 4, wherein theCIF130-binding domain binds to the CIF150/hTAF_(II)150-binding domain,wherein if the first fusion protein comprises a DNA binding domain, thenthe second fusion protein comprises a transcriptional activating domain,wherein if the first fusion protein comprises a transcriptionalactivating domain, then the second fusion protein comprises a DNAbinding domain, wherein the interaction of the first and second fusionproteins reconstitutes a sequence-specific transcription activatingfactor; and iii) a reporter gene comprising a DNA sequence to which theDNA binding domain specifically binds; and (b) measuring the expressionof the reporter gene, wherein a test compound which decreases theexpression of the reporter gene is a potential inducer of mitosis orcell cycle progression.